Search

Protein-protein and protein-water hydrogen bonding interactions play essential roles in the way a protein passes through the transition state during folding or unfolding, but the large number of these interactions in molecular dynamics (MD) simulations makes them difficult to analyze. Here, we introduce a state space representation and associated "rarity" measure to identify and quantify transition state passage (transit) events. Applying this representation to a long MD simulation trajectory that captured multiple folding and unfolding events of the GTT WW domain, a small protein often used as a model for the folding process, we identified three transition categories: Highway (faster), Meander (slower), and Ambiguous (intermediate). We developed data sonification and visualization tools to analyze hydrogen bond dynamics before, during, and after these transition events. By means of these tools, we were able to identify characteristic hydrogen bonding patterns associated with "Highway" versus "Meander" versus "Ambiguous" transitions and to design algorithms that can identify these same folding pathways and critical protein-water interactions directly from the data. Highly cooperative hydrogen bonding can either slow down or speed up transit. Furthermore, an analysis of protein-water hydrogen bond dynamics at the surface of WW domain shows an increase in hydrogen bond lifetime from folded to unfolded conformations with Ambiguous transitions as an outlier. In summary, hydrogen bond dynamics provide a direct window into the heterogeneity of transits, which can vary widely in duration (by a factor of 10) due to a complex energy landscape., Competing Interests: Competing interests statement:C.S. and K.J.H. are co-founders of Symbolic Sound Corporation and the developers of the Kyma software used for data analysis and sonification/visualization in this work.

Protein folding accuracy is fundamental to all cells. In spite of this, it is difficult to maintain the fidelity of protein synthesis and folding due to the fact that the implicit genetic and biochemical systems are inherently prone to error, which leads to the constant production of a certain amount of misfolded proteins. This problem is further compounded by genetic variation and the effects of environmental stress. To that end, the prediction of protein structures for tertiary protein structure analysis and prediction might be an ideal approach for the study of mutation effects in macromolecules and their complexes. With the development and accessibility to increasingly powerful computational systems, this type of study will enable a wide variety of opportunities for the creation of better-targeted peptide-based pharmacotherapy and prospects for precision medicine in future., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Studies of small proteins that exhibit noncooperative, gradual (un)folding can offer unique insights into the rarely accessible intermediate stages of the protein folding processes. Detailed experimental characterization of these intermediate states requires approaches that utilize multiple site-specific probes of the local structure. Isotopically edited infrared (IR) spectroscopy has emerged as a powerful methodology capable of providing such high-resolution structural information. Labeling of selected amide carbonyls with 13 C results in detectable side-bands of amide I' vibrations, which are sensitive to local conformation and/or solvent exposure without introducing any significant structural perturbation to the protein. Incorporation of isotopically labeled amino acids at specific positions can be achieved by the chemical synthesis of the studied proteins. We describe the basic procedures for synthesis of 13 C isotopically edited protein samples, experimental IR spectroscopic measurements and analysis of the site-specific equilibrium thermal unfolding of a small protein from the temperature-dependent IR data., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Computational simulations of protein folding can be used to interpret experimental folding results, to design new folding experiments, and to test the effects of mutations and small molecules on folding. However, whereas major experimental and computational progress has been made in understanding how small proteins fold, research on larger, multidomain proteins, which comprise the majority of proteins, is less advanced. Specifically, large proteins often fold via long-lived partially folded intermediates, whose structures, potentially toxic oligomerization, and interactions with cellular chaperones remain poorly understood. Molecular dynamics based folding simulations that rely on knowledge of the native structure can provide critical, detailed information on folding free energy landscapes, intermediates, and pathways. Further, increases in computational power and methodological advances have made folding simulations of large proteins practical and valuable. Here, using serpins that inhibit proteases as an example, we review native-centric methods for simulating the folding of large proteins. These synergistic approaches range from Gō and related structure-based models that can predict the effects of the native structure on folding to all-atom-based methods that include side-chain chemistry and can predict how disease-associated mutations may impact folding. The application of these computational approaches to serpins and other large proteins highlights the successes and limitations of current computational methods and underscores how computational results can be used to inform experiments. These powerful simulation approaches in combination with experiments can provide unique insights into how large proteins fold and misfold, expanding our ability to predict and manipulate protein folding., (© 2020 Gershenson et al.)

The majority of known bacteriophages have long tails that serve for bacterial target recognition and viral DNA delivery into the host. These structures form a tube from the viral capsid to the bacterial cell. The tube is formed primarily by a helical array of tail tube protein (TTP) subunits. In phages with a contractile tail, the TTP tube is surrounded by a sheath structure. Here, we report the first evidence that a phage TTP, gp17.1 of siphophage SPP1, self-assembles into long tubes in the absence of other viral proteins. gp17.1 does not exhibit a stable globular structure when monomeric in solution, even if it was confidently predicted to adopt the β-sandwich fold of phage λ TTP. However, Fourier transform infrared and nuclear magnetic resonance spectroscopy analyses showed that its β-sheet content increases significantly during tube assembly, suggesting that gp17.1 acquires a stable β-sandwich fold only after self-assembly. EM analyses revealed that the tube is formed by hexameric rings stacked helicoidally with the same organization and helical parameters found for the tail of SPP1 virions. These parameters were used to build a pseudo-atomic model of the TTP tube. The large loop spanning residues 40-56 is located on the inner surface of the tube, at the interface between adjacent monomers and hexamers. In line with our structural predictions, deletion of this loop hinders gp17.1 tube assembly in vitro and interferes with SPP1 tail assembly during phage particle morphogenesis in bacteria., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

To understand the relationship between protein sequence and structure, this work extends the knob-socket model in an investigation of β-sheet packing. Over a comprehensive set of β-sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four-residue packing cliques necessary to describe β-sheet packing were characterized. Both occur between two adjacent hydrogen bonded β-strands. First, defining the secondary structure packing within β-sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β-sheets, the knob-socket XY:H+B consists of a three-residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob-sockets are found: side-chain and main-chain sockets. The amino acid composition of the pockets and knob-sockets reveal the sequence specificity of β-sheet packing. For β-sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side-chain and main-chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β-sheet structure and provide an intuitive topological mapping of β-sheet packing., (© 2014 Wiley Periodicals, Inc.)

Targeted protein backbone modification can recreate tertiary structures reminiscent of folds found in nature on artificial scaffolds with improved biostability. Incorporation of altered monomers in such entities is typically limited to sites distant from the hydrophobic core to avoid potential disruptions to folding. This is limiting, as it is advantageous in some applications to incorporate artificial connectivity at buried sites. Here, we report an examination of protein backbone modification targeted specifically to hydrophobic core positions and its impacts on tertiary folded structure and fold stability. Different artificial monomer types are placed at core, core‐flanking, or solvent‐exposed positions in a compact three‐helix protein. Effects on structure and folding energetics are assessed by NMR spectroscopy and biophysical methods. Results show that artificial residues can be well accommodated in the hydrophobic core of a defined tertiary fold, with effects on stability only modestly larger than identical changes at solvent‐exposed sites. Collectively, these results provide new insights into folding behavior of protein‐like artificial chains as well as strategies for the design of such molecules. [ABSTRACT FROM AUTHOR]

A novel class of protein misfolding characterized by either the formation of non-native noncovalent lasso entanglements in the misfolded structure or loss of native entanglements has been predicted to exist and found circumstantial support through biochemical assays and limited-proteolysis mass spectrometry data. Here, we examine whether it is possible to design small molecule compounds that can bind to specific folding intermediates and thereby avoid these misfolded states in computer simulations under idealized conditions (perfect drug-binding specificity, zero promiscuity, and a smooth energy landscape). Studying two proteins, type III chloramphenicol acetyltransferase (CAT-III) and D-alanyl-D-alanine ligase B (DDLB), that were previously suggested to form soluble misfolded states through a mechanism involving a failure-to-form of native entanglements, we explore two different drug design strategies using coarse-grained structure-based models. The first strategy, in which the native entanglement is stabilized by drug binding, failed to decrease misfolding because it formed an alternative entanglement at a nearby region. The second strategy, in which a small molecule was designed to bind to a non-native tertiary structure and thereby destabilize the native entanglement, succeeded in decreasing misfolding and increasing the native state population. This strategy worked because destabilizing the entanglement loop provided more time for the threading segment to position itself correctly to be wrapped by the loop to form the native entanglement. Further, we computationally identified several FDA-approved drugs with the potential to bind these intermediate states and rescue misfolding in these proteins. This study suggests it is possible for small molecule drugs to prevent protein misfolding of this type. Author summary: A variety of diseases are caused by protein misfolding. Therefore, the recent evidence suggesting there is an entire unexplored class of protein misfolding that may be widespread opens the possibility they contribute to disease and may be therapeutic targets. Here, we bypass the question of what diseases such misfolding may contribute to and ask whether it is even possible to restore proper folding and function to proteins that misfold in this manner. We tried the most obvious strategy first: in the computer simulations we created a drug that binds to the folded, native entanglement. The rationale being that this would thermodynamically stabilize the native state relative to misfolded states, and thereby shift the population of molecules to the folded states. But thermodynamic reasoning neglects the folding pathways and kinetics connecting metastable states on the energy landscape, and this strategy had the unintended consequence of slowing down native state formation by stabilizing the entanglement loop without the threading segment piercing it, resulting in the loss of the native entanglement and formation of a non-native entanglement. Building on this observation, we took a different route, and designed a drug that would delay formation of the entanglement loop allowing more time for the threading segment to position itself and allow proper folding of the native entanglement. While this study was carried out using coarse-grained structure-based models, the results indicate it is possible in principle for drugs to be designed to avoid such misfolding and suggest our second design strategy is more likely to work in future experiments. [ABSTRACT FROM AUTHOR]

The crystal structure of the domain of unknown function family 507 protein from Aquifex aeolicus is reported (AaDUF507, UniProt O67633, 183 residues). The structure was determined in two space groups (C2221 and P3221) at 1.9 Å resolution. The phase problem was solved by molecular replacement using an AlphaFold model as the search model. AaDUF507 is a Y-shaped α-helical protein consisting of an anti-parallel 4-helix bundle base and two helical arms that extend 30-Å from the base. The two crystal structures differ by a 25° rigid body rotation of the C-terminal arm. The tertiary structure exhibits pseudo-twofold symmetry. The structural symmetry mirrors internal sequence similarity: residues 11–57 and 102–148 are 30% identical and 53% similar with an E-value of 0.002. In one of the structures, electron density for an unknown ligand, consistent with nicotinamide or similar molecule, may indicate a functional site. Docking calculations suggest potential ligand binding hot spots in the region between the helical arms. Structure-based query of the Protein Data Bank revealed no other protein with a similar tertiary structure, leading us to propose that AaDUF507 represents a new protein fold. [ABSTRACT FROM AUTHOR]

Background: The relationship between the sequence of a protein, its structure, and the resulting connection between its structure and function, is a foundational principle in biological science. Only recently has the computational prediction of protein structure based only on protein sequence been addressed effectively by AlphaFold, a neural network approach that can predict the majority of protein structures with X-ray crystallographic accuracy. A question that is now of acute relevance is the "inverse protein folding problem": predicting the sequence of a protein that folds into a specified structure. This will be of immense value in protein engineering and biotechnology, and will allow the design and expression of recombinant proteins that can, for instance, fold into specified structures as a scaffold for the attachment of recombinant antigens, or enzymes with modified or novel catalytic activities. Here we describe the development of SeqPredNN, a feed-forward neural network trained with X-ray crystallographic structures from the RCSB Protein Data Bank to predict the identity of amino acids in a protein structure using only the relative positions, orientations, and backbone dihedral angles of nearby residues. Results: We predict the sequence of a protein expected to fold into a specified structure and assess the accuracy of the prediction using both AlphaFold and RoseTTAFold to computationally generate the fold of the derived sequence. We show that the sequences predicted by SeqPredNN fold into a structure with a median TM-score of 0.638 when compared to the crystal structure according to AlphaFold predictions, yet these sequences are unique and only 28.4% identical to the sequence of the crystallized protein. Conclusions: We propose that SeqPredNN will be a valuable tool to generate proteins of defined structure for the design of novel biomaterials, pharmaceuticals, catalysts, and reporter systems. The low sequence identity of its predictions compared to the native sequence could prove useful for developing proteins with modified physical properties, such as water solubility and thermal stability. The speed and ease of use of SeqPredNN offers a significant advantage over physics-based protein design methods. [ABSTRACT FROM AUTHOR]

Although most globular proteins fold into a single stable structure, an increasing number have been shown to remodel their secondary and tertiary structures in response to cellular stimuli. State-of-the-art algorithms predict that these fold-switching proteins adopt only one stable structure, missing their functionally critical alternative folds. Why these algorithms predict a single fold is unclear, but all of them infer protein structure from coevolved amino acid pairs. Here, we hypothesize that coevolutionary signatures are being missed. Suspecting that single-fold variants could be masking these signatures, we developed an approach, called Alternative Contact Enhancement (ACE), to search both highly diverse protein superfamilies–composed of single-fold and fold-switching variants–and protein subfamilies with more fold-switching variants. ACE successfully revealed coevolution of amino acid pairs uniquely corresponding to both conformations of 56/56 fold-switching proteins from distinct families. Then, we used ACE-derived contacts to (1) predict two experimentally consistent conformations of a candidate protein with unsolved structure and (2) develop a blind prediction pipeline for fold-switching proteins. The discovery of widespread dual-fold coevolution indicates that fold-switching sequences have been preserved by natural selection, implying that their functionalities provide evolutionary advantage and paving the way for predictions of diverse protein structures from single sequences. Most globular proteins are selected to fold into one unique structure. Schafer and Porter demonstrate that some proteins are selected to assume two stable folds; they leverage this information to predict two structures from one sequence. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of RNA molecules plays a critical role in a wide range of cellular processes encompassing functions from riboswitches to epigenetic regulation. These RNA structures are incredibly dynamic and can indeed be described aptly as an ensemble of structures that shifts in distribution depending on different cellular conditions. Thus, the computational prediction of RNA structure poses a unique challenge, even as computational protein folding has seen great advances. In this review, we focus on a variety of machine learning-based methods that have been developed to predict RNA molecules' secondary structure, as well as more complex tertiary structures. We survey commonly used modeling strategies, and how many are inspired by or incorporate thermodynamic principles. We discuss the shortcomings that various design decisions entail and propose future directions that could build off these methods to yield more robust, accurate RNA structure predictions. [ABSTRACT FROM AUTHOR]

A comprehensive understanding of the interfacial behaviors of biomolecules holds great significance in the development of biomaterials and biosensing technologies. In this work, we used discontinuous molecular dynamics (DMD) simulations and graphic contrastive learning analysis to study the adsorption of ubiquitin protein on a graphene surface. Our high-throughput DMD simulations can explore the whole protein adsorption process including the protein structural evolution with sufficient accuracy. Contrastive learning was employed to train a protein contact map feature extractor aiming at generating contact map feature vectors. Subsequently, these features were grouped using the k-means clustering algorithm to identify the protein structural transition stages throughout the adsorption process. The machine learning analysis can illustrate the dynamics of protein structural changes, including the pathway and the rate-limiting step. Our study indicated that the protein–graphene surface hydrophobic interactions and the π–π stacking were crucial to the seven-stage adsorption process. Upon adsorption, the secondary structure and tertiary structure of ubiquitin disintegrated. The unfolding stages obtained by contrastive learning-based algorithm were not only consistent with the detailed analyses of protein structures but also provided more hidden information about the transition states and pathway of protein adsorption process and structural dynamics. Our combination of efficient DMD simulations and machine learning analysis could be a valuable approach to studying the interfacial behaviors of biomolecules. [ABSTRACT FROM AUTHOR]

The tick-transmitted disease bovine babesiosis causes significant economic losses in many countries around the world. Current control methods include modified live-attenuated vaccines that have limited efficacy. Recombinant proteins could provide effective, safe, and low-cost alternative vaccines. We compared the expression of the Babesia bovis thrombospondin-related anonymous protein (TRAP) family from parasites in bovine blood, in vitro induced sexual stages, and kinetes from tick hemolymph. Quantitative PCR showed that in blood and sexual stages, TRAP3 was highly transcribed as compared to the other TRAPs. In contrast, the TRAP1 gene was highly transcribed in kinetes as compared to the other TRAPs. Fixed immunofluorescence assays showed that TRAP2, 3, and 4 proteins were expressed by both blood and sexual stages. Conversely, TRAP1 protein, undetected on blood and induced sexual stages, was the only family member expressed by kinetes. Live IFA revealed that TRAP2, 3, and 4 proteins were expressed on the surface of both B. bovis blood and sexual stages. Modeling of B. bovis TRAP1 and TRAP4 tertiary structure demonstrated both proteins folded the metal-ion-dependent adhesion site (MIDAS) domain structure of Plasmodium TRAP. In conclusion, TRAP proteins may serve as potential vaccine targets to prevent infection of bovine and ticks with B. bovis essential for controlling the spread of bovine babesiosis. [ABSTRACT FROM AUTHOR]

Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/β-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (Ht DLH). The crystal structure of the Ht DLH, solved at a resolution of about 1.67 Å, exhibits a canonical α/β-hydrolase fold formed by eight β-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The Ht DLH forms stable homodimers in solution. Functional studies showed that Ht DLH has the expected esterase activity over esters with short carbon chains, such as p- nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 °C. Furthermore, Ht DLH maintains more than 50 % of its activity even after incubation at 90 °C for 16 h. Interestingly, Ht DLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that Ht DLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments. • Ht DLH solved at a resolution of 1.67 Å exhibits a canonical α/β-hydrolase fold. • Ht DLH exhibits a non-canonical PAZyme catalytic triad. • Ht DLH exhibits activity towards PET monomers, and aliphatic and aromatic esters. • Ht DLH is a candidate with potential application in PET depolymerization. [ABSTRACT FROM AUTHOR]

Protein folding in the cell is largely a co-translational process occurring during protein synthesis on the ribosome. It has become evident that co-translational folding is characteristic to almost every protein in the cell of pro- and eukaryotic origin that are single and multidomain, single and multisubunit, cytosolic, secretory and membrane. Co-translational protein folding begins very early during the process of polypeptide chain synthesis on the ribosome, with some secondary structure elements forming inside the ribosomal tunnel and some tertiary structures forming inside the vestibule (lower/wider) region of the ribosomal exit tunnel. However, many details of co-translational folding remains incompletely understood. New data show that folding of a β-barrel protein begins with formation of an α-helix inside the ribosome that rearranges into a β-hairpin structure as the growing peptide reaches the wider/vestibule region of the exit tunnel. While it was previously suggested that such scenario can take place on the ribosome, the new data provide the first experimental evidence in support of this notion. [ABSTRACT FROM AUTHOR]

The pioneering experiments of Anfinsen on the oxidative folding of RNase have been revisited discovering some details, which update the statement of his dogma and shed new light on the leading role of the correct disulfide in the attainment of the native structure. CD analysis, mass spectrometry, fluorescence spectroscopy and enzyme activity indicate that native disulfides drive the formation of the secondary and tertiary structures that cannot be entirely formed in their absence. This opposes a common opinion that these structures are first formed and then stabilized by the native disulfides. Our results also indicate that a spontaneous re-oxidation of a reduced RNase cannot produce a complete recovery of activity, as described by many textbooks; this can be obtained only in the presence of a reshuffling solution such as GSH/GSSG. [ABSTRACT FROM AUTHOR]

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability. [ABSTRACT FROM AUTHOR]

The folding dynamics of proteins is a primary area of interest in protein science. We carried out topological data analysis (TDA) of the folding process of HP35(nle-nle), a double-mutant of the villin headpiece subdomain. Using persistent homology and non-negative matrix factorization, we reduced the dimension of protein structure and investigated the flow in the reduced space. We found this protein has two folding paths, distinguished by the pairings of inter-helix residues. Our analysis showed the excellent performance of TDA in capturing the formation of tertiary structure. [ABSTRACT FROM AUTHOR]

Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to "buried" domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser339 and Ser340 abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond "simple" generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes. [ABSTRACT FROM AUTHOR]

The problem of linear polypeptide chain folding into a unique tertiary structure is one of the fundamental scientific challenges. The process of folding cannot be fully understood without its biological context, especially for big multidomain and multisubunit proteins. The principal features of biosynthetic folding are co-translational folding of growing nascent polypeptide chains and involvement of molecular chaperones in the process. The review summarizes available data on the early events of nascent chain folding, as well as on later advanced steps, including formation of elements of native structure. The relationship between the non-uniformity of translation rate and folding of the growing polypeptide is discussed. The results of studies on the effect of biosynthetic folding features on the parameters of folding as a physical process, its kinetics and mechanisms, are presented. Current understanding and hypotheses on the relationship of biosynthetic folding with the fundamental physical parameters and current views on polypeptide folding in the context of energy landscapes are discussed. [ABSTRACT FROM AUTHOR]

In a cell, the folding of a protein molecule into tertiary structure can begin while it is synthesized by the ribosome. The rate at which individual amino acids are incorporated into the elongating nascent chain has been shown to affect the likelihood that proteins will populate their folded state, indicating that co-translational protein folding is a far from equilibrium process. Developing a theoretical framework to accurately describe this process is, therefore, crucial for advancing our understanding of how proteins acquire their functional conformation in living cells. Current state-of-the-art computational approaches, such as molecular dynamics simulations, are very demanding in terms of the required computer resources, making the simulation of co-translational protein folding difficult. Here, we overcome this limitation by introducing an efficient approach that predicts the effects that variable codon translation rates have on co-translational folding pathways. Our approach is based on Markov chains. By using as an input a relatively small number of molecular dynamics simulations, it allows for the computation of the probability that a nascent protein is in any state as a function of the translation rate of individual codons along a mRNA's open reading frame. Due to its computational efficiency and favorable scalability with the complexity of the folding mechanism, this approach could enable proteome-wide computational studies of the influence of translation dynamics on co-translational folding. [ABSTRACT FROM AUTHOR]

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here, the authors employ an approach that uses cryo-EM single particle analysis to track the number and distribution of nucleotides bound to each subunit in the homo-oligomeric MmCpn archaeal chaperonin complex and observe that ATP binds in a statistically random manner to MmCpn both within a ring and across the rings, which shows that there is no cooperativity in ATP binding to archaeal group II chaperonins under the conditions used in this study. [ABSTRACT FROM AUTHOR]

The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure–function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore-forming peptide into a selective pore-former for different lipid types. A library of 2916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization. [ABSTRACT FROM AUTHOR]

Previously, the structure elements of dihydrofolate reductase (DHFR) were determined using comprehensive Ala-insertion mutation analysis, which is assumed to be a kind of protein "building blocks." It is hypothesized that our comprehension of the structure elements could lead to understanding how an amino acid sequence dictates its tertiary structure. However, the comprehensive Ala-insertion mutation analysis is a time- and cost-consuming process and only a set of the DHFR structure elements have been reported so far. Therefore, developing a computational method to predict structure elements is an urgent necessity. We focused on intramolecular residue-residue contacts to predict the structure elements. We introduced a simple and effective parameter: the overlapped contact volume (CV) among the residues and calculated the CV along the DHFR sequence using the crystal structure. Our results indicate that the CV profile can recapitulate its precipitate ratio profile, which was used to define the structure elements in the Ala-insertion mutation analysis. The CV profile allowed us to predict structure elements like the experimentally determined structure elements. The strong correlation between the CV and precipitate ratio profiles indicates the importance of the intramolecular residue-residue contact in maintaining the tertiary structure. Additionally, the CVs between the structure elements are considerably more than those between a structure element and a linker or two linkers, indicating that the structure elements play a fundamental role in increasing the intramolecular adhesion. Thus, we propose that the structure elements can be considered a type of "building blocks" that maintain and dictate the tertiary structures of proteins. [ABSTRACT FROM AUTHOR]

Metallo-β-lactamases (MBLs) hydrolyze a wide range of β-lactam antibiotics. While all MBLs share a common αβ/βα-fold, there are many other proteins with the same folding pattern that exhibit different enzymatic activities. These enzymes, together with MBLs, form the MBL superfamily. Thermotoga maritima tRNase Z, a tRNA 3′ processing endoribonuclease of MBL-superfamily, and IMP-1, a clinically isolated MBL, showed a striking similarity in tertiary structure, despite low sequence homology. IMP-1 hydrolyzed both total cellular RNA and synthetic small unstructured RNAs. IMP-1 also hydrolyzed pre-tRNA, but its cleavage site was different from those of T. maritima tRNase Z and human tRNase Z long form, indicating a key difference in substrate recognition. Single-turnover kinetic assays suggested that substrate-binding affinity of T. maritima tRNase Z is much higher than that of IMP-1. [ABSTRACT FROM AUTHOR]

Simian betaretrovirus serotype-2 (SRV-2) is an important pathogenic agent in Asian macaques. It is a potential confounding variable in biomedical research. SRV-2 also provides a valuable viral model compared to other retroviruses which can be used for understanding many aspects of retroviral-host interactions and immunosuppression, infection mechanism, retroviral structure, antiretroviral and vaccine development. In this study, we isolated the gene encoding reverse transcriptase enzyme (RT) of SRV-2 that infected Indonesian cynomolgus monkey (Mf ET1006) and predicted the three dimensional structure model using the iterative threading assembly refinement (I-TASSER) computational programme. This SRV-2 RT Mf ET1006 consisted of 547 amino acids at nucleotide position 3284-4925 of whole genome SRV-2. The polymerase active site located in the finger/palm subdomain characterised by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165 and Asp166. We estimated that this SRV-2 RT Mf ET1006 structure has the accuracy of template modelling score (TM-score 0.90 ± 0.06) and root mean square deviation (RMSD) 4.7 ± 3.1Å, indicating that this model can be trusted and the accuracy can be seen from the appearance of protein folding in tertiary structure. The superpositionings between SRV-2 RT Mf ET1006 and Human Immunodeficiency Virus-1 (HIV-1) RT were performed to predict the structural in details and to optimise the best fits for illustrations. This SRV-2 RT Mf ET1006 structure model has the highest homology to HIV-1 RT (2B6A.pdb) with estimated accuracy at TM-score 0.911, RMSD 1.85 Å, and coverage of 0.953. This preliminary study of SRV-2 RT Mf ET1006 structure modelling is intriguing and provide some information to explore the molecular characteristic and biochemical mechanism of this enzyme. [ABSTRACT FROM AUTHOR]

To understand protein folding problem under physiological condition, usually taken as dilute aqueous buffer at pH 7.0 and 25 °C, knowledge of properties of folding intermediates is important, such as molten globule (MG). We observed that polyethylene glycol 400 Da (PEG 400) induces molten globule state conformation in cytochrome c at pH 7.0 and 25 °C. This PEG-induced MG state has: (i) native tertiary structure partially perturbed, (ii) unperturbed native secondary structure, (iii) newly exposed hydrophobic patches, and (iv) has 1.58 times more hydrodynamic volume than that of the native protein. Isothermal titration calorimetry and docking studies showed specific binding between PEG 400 and cytochrome c. The study delineates that PEG-protein interactions are more complex than the excluded-volume. The soft interactions need to be seriously studied in crowding milieu that leads to destabilization of protein and overcome stabilizing exclusion volume effect. This study not only can help in unraveling the mystery of steps involved in the proper folding of proteins to solve the massively complicated problems of protein folding but also provides novel insights towards importance of structural change in proteins inside cell where intermediate states of protein import-export easily via membranes rather than native form of proteins. Unlabelled Image • PEG 400 is a low-level toxic ingredient in drug formulations to augment the solubility and bioavailability of weakly water-soluble drugs. • The PEG 400 yields molten globule of holo-cytochrome c , characterized at physiological pH and 25 oC. • The PEG 400 interacts with cytochrome c and its prosthetic group by soft interactions that lead the structural change in the protein. • The exclusion volume effect is surmounted by soft interactions which leads formation of stabilized state of cytochrome c (molten globule). • Importance of formation of molten globule lies in protein import-export easily via membranes rather than native form of proteins. [ABSTRACT FROM AUTHOR]

The interpretation of experimental studies of co-translational protein folding often benefits from the use of computational methods that seek to model or simulate the nascent chain and its interactions with the ribosome. Building realistic 3D models of ribosome-nascent chain (RNC) constructs often requires expert knowledge, so to circumvent this issue, we describe here AutoRNC, an automated modeling program capable of constructing large numbers of plausible atomic models of RNCs within minutes. AutoRNC takes input from the user specifying any regions of the nascent chain that contain secondary or tertiary structure and attempts to build conformations compatible with those specifications—and with the constraints imposed by the ribosome—by sampling and progressively piecing together dipeptide conformations extracted from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB). Despite using only modest computational resources, we show here that AutoRNC can build plausible conformations for a wide range of RNC constructs for which experimental data have already been reported. [Display omitted] • AutoRNC allows atomic models of ribosome-nascent chain complexes to be built rapidly • Nascent chains can be built with any combination of secondary and tertiary structure • In favorable cases, thousands of nascent chain models can be built in minutes McDonnell and Elcock report a fast but simple computational protocol for building atomic models of nascent protein chains as they are synthesized by the ribosome. Models can be built with any combination of secondary and tertiary structure that is compatible with the steric restrictions imposed by the ribosome exit tunnel. [ABSTRACT FROM AUTHOR]

Protein fibrils are of great interest due to their involvement in several pathologies and their roles in the degradation of many therapeutic protein products. Fibrils share highly similar secondary structural motifs across different proteins and applied stress conditions. However, fibril morphology differs according to the surrounding conditions, with aromatic and hydrophobic amino acids playing important roles in mature fibril formation. In this study, we use Raman microscopy, by means of the aromatic amino acids in insulin molecules as markers, to probe for tertiary structure differences within fibrils. We compared 2 different fibril types, linear fibril bundles and spherulites. Generation of linear fibril bundles was undertaken in an acetic acid–containing formulation, whereas spherulites were generated in a hydrochloric acid–containing formulation. The Raman intensities of tyrosine and phenylalanine side chains suggest that there are significant differences between the fibril bundles. The findings suggest that the insulin components of the fibril strands are not arranged identically in the 2 fibril types and that this gives rise to differences in their tertiary structures. Overall, the work indicates that the physicochemical properties of fibril structures can be altered by changing the formulation and that these alterations can be monitored by Raman spectroscopy. [ABSTRACT FROM AUTHOR]

Most proteins exist with multiple domains in cells for cooperative functionality. However, structural biology and protein folding methods are often optimized for single-domain structures, resulting in a rapidly growing gap between the improved capability for tertiary structure determination and high demand for multidomain structure models. We have developed a pipeline, termed DEMO, for constructing multidomain protein structures by docking-based domain assembly simulations, with interdomain orientations determined by the distance profiles from analogous templates as detected through domain-level structure alignments. The pipeline was tested on a comprehensive benchmark set of 356 proteins consisting of 2-7 continuous and discontinuous domains, for which DEMO generated models with correct global fold (TM-score > 0.5) for 86% of cases with continuous domains and for 100% of cases with discontinuous domain structures, starting from randomly oriented target-domain structures. DEMO was also applied to reassemble multidomain targets in the CASP12 and CASP13 experiments using domain structures excised from the top server predictions, where the full-length DEMO models showed a significantly improved quality over the original server models. Finally, sparse restraints of mass spectrometry-generated cross-linking data and cryo-EM density maps are incorporated into DEMO, resulting in improvements in the average TM-score by 6.3% and 12.5%, respectively. The results demonstrate an efficient approach to assembling multidomain structures, which can be easily used for automated, genome-scale multidomain protein structure assembly. [ABSTRACT FROM AUTHOR]

Sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis , is a novel model system for membrane protein folding studies. Recently, the SDS-denatured states and the kinetics for reversible unfolding of pSRII have been investigated, opening the door to the first detailed characterisation of denatured states of a membrane protein by solution-state nuclear magnetic resonance (NMR) using uniformly 15N-labelled pSRII. SDS denaturation and acid denaturation of pSRII both lead to fraying of helix ends but otherwise small structural changes in the transmembrane domain, consistent with little changes in secondary structure and disruption of the retinal-binding pocket and tertiary structure. Widespread changes in the backbone amide dynamics are detected in the form of line broadening, indicative of μs-to-ms timescale conformational exchange in the transmembrane region. Detailed analysis of chemical shift and intensity changes lead to high-resolution molecular insights on structural and dynamics changes in SDS- and acid-denatured pSRII, thus highlighting differences in the unfolding pathways under the two different denaturing conditions. These results will form the foundation for furthering our understanding on the folding and unfolding pathways of retinal-binding proteins and membrane proteins in general, and also for investigating the importance of ligand-binding in the folding pathways of other ligand-binding membrane proteins, such as GPCRs. Unlabelled Image • We report detailed NMR studies on a denatured polytopic α-helical membrane protein • SDS- and acid-denatured pSRII experience small structural changes in the TM domains • Denatured pSRII undergoes widespread μs-to-ms timescale conformational exchange • High-resolution structural differences between SDS vs. acid denaturation are shown • Retinal influences the exchange equilibria amongst different conformations of pSRII [ABSTRACT FROM AUTHOR]

[Display omitted] • New proteins emerge in all organisms but little is known regarding their structure. • Putative new small proteins were identified in Apilactobacillus kunkeii. • These "smORF" proteins display cooperative folding and are marginally stable. • The smORF proteins are predominantly alpha helical. • smORFs may evolve to converge on existing protein folds. The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0–3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event. [ABSTRACT FROM AUTHOR]

During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the exit port where it emerges into the cytoplasm. The ribosome defines the unique energy landscape of the pioneering round of protein folding. The spatial confinement and the interactions of the nascent peptide with the tunnel walls facilitate formation of secondary structures, such as α-helices. The vectorial nature of protein folding inside the tunnel favors local intra- and inter-molecular interactions, thereby inducing cotranslational folding intermediates that do not form upon protein refolding in solution. Tertiary structures start to fold in the lower part of the tunnel, where interactions with the ribosome destabilize native protein folds. The present review summarizes the recent progress in understanding the driving forces of nascent protein folding inside the tunnel and at the surface of the ribosome. [ABSTRACT FROM AUTHOR]

Proteins are essential biological macromolecules associated with a variety of physiological functions. In this article, the composition, conformational features, and general principles that govern protein folding are presented. [ABSTRACT FROM AUTHOR]

Protein design holds promise for applications such as the control of cells, therapeutics, new enzymes and protein-based materials. Recently, there has been progress in rational design of protein molecules, and a lot of attempts have been made to create proteins with functions of our interests. The key to the progress is the development of methods for controlling desired protein tertiary structures with atomic-level accuracy. A theory for protein folding, the consistency principle, proposed by Nobuhiro Go in 1983, was a compass for the development. Anfinsen hypothesized that proteins fold into the free energy minimum structures, but Go further considered that local and non-local interactions in the free energy minimum structures are consistent with each other. Guided by the principle, we proposed a set of rules for designing ideal protein structures stabilized by consistent local and non-local interactions. The rules made possible designs of amino acid sequences with funnel-shaped energy landscapes toward our desired target structures. So far, various protein structures have been created using the rules, which demonstrates significance of our rules as intended. In this review, we briefly describe how the consistency principle impacts on our efforts for developing the design technology. [ABSTRACT FROM AUTHOR]

The prospect of recreating the complex structural hierarchy of protein folding in synthetic oligomers with backbones that are artificial in covalent structure ("foldamers") has long fascinated chemists. Foldamers offer complex functions from biostable scaffolds and have found widespread applications in fields from biomedical to materials science. Most precedent has focused on isolated secondary structures or their assemblies. In considering the goal of complex protein-like tertiary folding patterns, a key barrier became apparent. How does one design a backbone with covalent connectivity and a sequence of side-chain functional groups that will support defined intramolecular packing of multiple artificial secondary structures? Two developments were key to overcoming this challenge. First was the recognition of the power of blending α-amino acid residues with monomers differing in backbone connectivity to create "heterogeneous-backbone" foldamers. Second was the finding that replacing some of the natural α-residues in a biological sequence with artificial-backbone variants can result in a mimic that retains both the fold and function of the native sequence and, in some cases, gains advantageous characteristics. Taken together, these precedents lead to a view of a protein as chemical entity having two orthogonal sequences: a sequence of side-chain functional groups and a separate sequence of backbone units displaying those functional groups. In this Account, we describe our lab's work over the last ~10 years to leverage the above concept of protein sequence duality in order to develop design principles for constructing heterogeneous-backbone foldamers that adopt complex protein-like tertiary folds. Fundamental to the approach is the utilization of a variety of artificial building blocks (e.g., d-α-residues, Cα-Me-α-residues, N-Me-α-residues, β-residues, γ-residues, δ-residues, polymer segments) in concert, replacing a fraction of α-residues in a given prototype sequence. We provide an overview of the state-of-the-art in terms of design principles for choosing substitutions based on consideration of local secondary structure and retention of key side-chain functional groups. We survey high-resolution structures of backbone-modified proteins to illustrate how diverse artificial moieties are accommodated in tertiary fold contexts. We detail efforts to elucidate how backbone alteration impacts folding thermodynamics and describe how such data informs the development of improved design rules. Collectively, a growing body of results by our lab and others spanning multiple protein systems suggests there is a great deal of plasticity with respect to the backbone chemical structures upon which sequence-encoded tertiary folds can manifest. Moreover, these efforts suggest sequence-guided backbone alteration as a broadly applicable strategy for generating foldamers with complex tertiary folding patterns. We conclude by offering some perspective regarding the near future of this field, in terms of unanswered questions, technological needs, and opportunities for new areas of inquiry. [ABSTRACT FROM AUTHOR]

RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The three-dimensional structure forms a parallel β-roll, where β-strands of two parallel sheets are connected by calcium-binding linkers in such a way that a right-handed spiral is built. The Ca2+ ions are an integral part of the structure, which cannot form without them. The structural determinants of this unique architecture will be reviewed with its conservations and variations together with the implication for secretion and folding of these proteins. The general purpose of the RTX domains appears to act as an internal chaperone that keeps the polypeptide unfolded in the calcium-deprived cytosol and triggers folding in the calcium-rich extracellular medium. A rather recent addition to the structural biology of the RTX toxin is a variant occurring in a large RTX adhesin, where this non-canonical β-roll binds to ice and diatoms.

Excitons in light-harvesting complexes are known to significantly improve solar-energy harnessing. Here we demonstrate photosynthetic excitons at super-physiological temperatures reaching 60-80 °C in different species of mesophilic photosynthetic bacteria. It is shown that the survival of light-harvesting excitons in the peripheral LH2 antennae is restricted by thermal decomposition of the pigment-protein complex rather than by any intrinsic property of excitons. The regular spatial organization of the bacteriochlorophyll a pigments supporting excitons in this complex is lost upon the temperature-induced breakdown of its tertiary structure. Secondary structures of the complexes survive even higher temperatures. The discovered pivotal role of the protein scaffold in the stabilization of excitons comprises an important aspect of structure-function relationship in biology. These results also intimately entangle the fundamental issues of quantum mechanical concepts in biology and in the folding of proteins. [ABSTRACT FROM AUTHOR]

Prediction of protein tertiary structures from amino acid sequence and understanding the mechanisms of how proteins fold, collectively known as “the protein folding problem,” has been a grand challenge in molecular biology for over half a century. Theories have been developed that provide us with an unprecedented understanding of protein folding mechanisms. However, computational simulation of protein folding is still difficult, and prediction of protein tertiary structure from amino acid sequence is an unsolved problem. Progress toward a satisfying solution has been slow due to challenges in sampling the vast conformational space and deriving sufficiently accurate energy functions. Nevertheless, several techniques and algorithms have been adopted to overcome these challenges, and the last two decades have seen exciting advances in enhanced sampling algorithms, computational power and tertiary structure prediction methodologies. This review aims at summarizing these computational techniques, specifically conformational sampling algorithms and energy approximations that have been frequently used to study protein-folding mechanisms or tode novopredict protein tertiary structures. We hope that this review can serve as an overview on how the protein-folding problem can be studied computationally and, in cases where experimental approaches are prohibitive, help the researcher choose the most relevant computational approach for the problem at hand. We conclude with a summary of current challenges faced and an outlook on potential future directions. [ABSTRACT FROM PUBLISHER]

Heme binding by proteins and protein-protein complexation are the processes strongly related to the biological activity of proteins. The mechanism of these processes has not been still recognised. These phenomena are presented using haemoglobin as the example. Half of the mature haemoglobin (one a-chain and one ß-chain) treated as a dissociation step in haemoglobin degradation reveals a specific change in heme binding after dissociation. This phenomenon is the object of analysis that interprets the structure of both complexes (tetramer and dimer) with respect to their hydrophobic core structure. The results suggest the higher stability of the complex in the form of one a-chain and one β-chain with respect to the hydrophobic core. [ABSTRACT FROM AUTHOR]

Background Three NPXY motifs and one FERM domain in CCM1 makes it a versatile scaffold protein for tethering the signaling components together within the CCM signaling complex (CSC). The cellular role of CCM1 protein remains inadequately expounded. Both phosphotyrosine binding (PTB) and pleckstrin homology (PH) domains were recognized as structurally related but functionally distinct domains. Methods By utilizing molecular cloning, protein binding assays and RT-qPCR to identify novel cellular partners of CCM1 and its cellular expression patterns; by screening candidate PTB/PH proteins and subsequently structurally simulation in combining with current X-ray crystallography and NMR data to defined the essential structure of PTB/PH domain for NPXY-binding and the relationship among PTB, PH and FERM domain(s). Results We identified a group of 28 novel cellular partners of CCM1, all of which contain either PTB or PH domain(s), and developed a novel classification system for these PTB/PH proteins based on their relationship with different NPXY motifs of CCM1. Our results demonstrated that CCM1 has a wide spectrum of binding to different PTB/PH proteins and perpetuates their specificity to interact with certain PTB/PH domains through selective combination of three NPXY motifs. We also demonstrated that CCM1 can be assembled into oligomers through intermolecular interaction between its F3 lobe in FERM domain and one of the three NPXY motifs. Despite being embedded in FERM domain as F3 lobe, F3 module acts as a fully functional PH domain to interact with NPXY motif. The most salient feature of the study was that both PTB and PH domains are structurally and functionally comparable, suggesting that PTB domain is likely evolved from PH domain with polymorphic structural additions at its N-terminus. Conclusions A new β1A-strand of the PTB domain was discovered and new minimum structural requirement of PTB/PH domain for NPXY motif-binding was determined. Based on our data, a novel theory of structure, function and relationship of PTB, PH and FERM domains has been proposed, which extends the importance of the NPXY-PTB/PH interaction on the CSC signaling and/or other cell receptors with great potential pointing to new therapeutic strategies. General significance The study provides new insight into the structural characteristics of PTB/PH domains, essential structural elements of PTB/PH domain required for NPXY motif-binding, and function and relationship among PTB, PH and FERM domains. [ABSTRACT FROM AUTHOR]

ABSTRACT [ABSTRACT FROM AUTHOR]

• Trehalose enhances chaperone activity of both α- casein and β-casein proteins at all concentrations. • Significant loss of secondary and tertiary structure for both the IDPs in presence of trehalose. • Significant increase in fluorescence with enhanced exposure of hydrophobic patches for both IDPs in presence of trehalose. • Increased proteolytic digestion susceptibility of both the IDPs in presence of trehalose. Intrinsically disordered proteins (IDPs) are being currently explored for their role in wide range of essential cellular processes especially signalling, cell cycle, transcription, translation and several disease processes like neurodegeneration, cancer, systemic amyloidosis etc. However, as compared to ordered proteins, their high structural dynamics makes them quite sensitive to the changing cellular environment such as changes in the level of cellular metabolites. Since previous studies have shown the ability of trehalose (an important small molecule metabolite) to affect protein folding and formation of high order oligomers, we aim to elucidate the effect of trehalose on the structural–functional elasticity of intrinsically disordered α- and β-casein proteins. We observed that trehalose enhances disorderliness resulting in increased chaperone activity of both the casein proteins. Considering the role of co-solutes in cellular proteostasis, the present study indicates that trehalose acts not only as an osmoprotectant, but also a key regulator of functional activity of various IDPs. [ABSTRACT FROM AUTHOR]

Folding of proteins usually involves intermediates, of which an important type is the molten globule (MG). MGs are ensembles of interconverting conformers that contain (non-)native secondary structure and lack the tightly packed tertiary structure of natively folded globular proteins. Whereas MGs of various purified proteins have been probed to date, no data are available on their presence and/or effect during protein synthesis. To study whether MGs arise during translation, we use ribosomenascent chain (RNC) complexes of the electron transfer protein flavodoxin. Full-length isolated flavodoxin, which contains a non-covalently bound flavin mononucleotide (FMN) as cofactor, acquires its native/ parallel topology via a folding mechanism that contains an off-pathway intermediate with molten globular characteristics. Extensive population of this MG state occurs at physiological ionic strength for apoflavodoxin variant F44Y, in which a phenylalanine at position 44 is changed to a tyrosine. Here, we show for the first time that ascertaining the binding rate of FMN as a function of ionic strength can be used as a tool to determine the presence of the off-pathwayMGon the ribosome. Application of this methodology to F44Y apoflavodoxin RNCs shows that at physiological ionic strength the ribosome influences formation of the off-pathway MG and forces the nascent chain toward the native state. [ABSTRACT FROM AUTHOR]

Protein folding, tagged as a grand challenge/NP hard problem, has been an open area of research in diverse fields. Exploring folding from the configurational volume perspective in terms of phi (ϕ), psi(ψ) backbone dihedral angles, we asked ourselves a fundamental question as to when do the neighborhood effects on the allowed ϕ, ψ values of each residue take over to assure convergence of proteins to their observed unique tertiary structures. A mapping of the higher order steric correlations beyond Ramachandran plots from ∼43612 protein structures comprising ∼26.5 million amino acid residues reveals that conformational restrictions on allowed ϕ, ψ values of each amino acid residue due to the N and C terminal neighbors – essentially a consideration of the sterically allowed regions of a tripeptide - ensure convergence of the configurational volume. [ABSTRACT FROM AUTHOR]

In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially misfolded intermediates aggregate via the interaction with the complementary intermediates and consequently enhance oligomers formation that grows into fibrils and proto-fibrils. The amyloid fibrils for example, accumulate in the brain and central nervous system (CNS) as amyloid deposits in the Parkinson’s disease (PD), Alzheimer’s disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other neurodegenerative diseases. Treatment of such misfolding maladies is considered as one of the most important challenges of the 21st century. Currently, several treatments strategies have been and are being discovered. These therapeutic interventions partly reversed or prevented the pathological state. More recently, a new approach was discovered, which employs nanobodies that targets multisteps in fibril formation pathway that may possibly completely cure these misfolding diseases. Keeping the above views in mind in the current review, we have comprehensively discussed the different mechanisms underlying protein misfolding thereby leading to diseases conditions and their therapeutic interventions. [ABSTRACT FROM AUTHOR]

Can the structures of small to medium-sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion-mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β-lactoglobulin, which are prototypical examples of helical and β-sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved. [ABSTRACT FROM AUTHOR]