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Fluorescent unnatural amino acids give biochemists, biophysicists, and bioengineers new ways to probe the properties of proteins and peptides. Here, the synthesis of acridon-2-ylalanine (Acd) is optimized for large-scale production to enable ribosomal incorporation through genetic code expansion (GCE), and fluorenylmethoxycarbonyl (Fmoc)-protected Acd is prepared for solid-phase peptide synthesis (SPPS). We demonstrate the utility of Acd in several applications: first, Acd quenching by Tyr is used in the design of fluorescent protease sensors made by SPPS. Second, we demonstrate Acd incorporation into a lanthanide-binding peptide that is generated either by GCE or by SPPS and demonstrate the utility of Acd for sensitizing the emission of Eu 3+ . Finally, Acd is inserted into the intrinsically disordered protein, α-synuclein, using GCE and used to study ion binding and aggregation.

The prediction of peptide amyloidogenesis is a challenging problem in the field of protein folding. Large language models, such as the ProtBERT model, have recently emerged as powerful tools in analyzing protein sequences for applications, such as predicting protein structure and function. In this article, we describe the use of a semisupervised and fine-tuned ProtBERT model to predict peptide amyloidogenesis from sequences alone. Our approach, which we call AggBERT, achieved state-of-the-art performance, demonstrating the potential for large language models to improve the accuracy and speed of amyloid fibril prediction over simple heuristics or structure-based approaches. This work highlights the transformative potential of machine learning and large language models in the fields of chemical biology and biomedicine.

Abnormal α-synuclein (α-syn) aggregation characterizes α-synucleinopathies, including Parkinson's disease (PD) and multiple system atrophy (MSA). However, no suitable positron emission tomography (PET) radiotracer for imaging α-syn in PD and MSA exists currently. Our structure-activity relationship studies identified 4-methoxy- N -(4-(3-(pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)benzamide ( 4i ) as a PET radiotracer candidate for imaging α-syn. In vitro assays revealed high binding of 4i to recombinant α-syn fibrils (inhibition constant ( K i ) = 6.1 nM) and low affinity for amyloid beta (Aβ) fibrils in Alzheimer's disease (AD) homogenates. However, [ 3 H]4i also exhibited high specific binding to AD, progressive supranuclear palsy, and corticobasal degeneration tissues as well as PD and MSA tissues, suggesting notable affinity to tau. Nevertheless, the specific binding to pathologic α-syn aggregates in MSA post-mortem brain tissues was significantly higher than in PD tissues. This finding demonstrated the potential use of [ 11 C]4i as a PET tracer for imaging α-syn in MSA patients. Nonhuman primate PET studies confirmed good brain uptake and rapid washout for [ 11 C]4i .

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases. A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson's disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of αS have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here, we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human αS, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, αS remains disordered when in complex with hNatB. We further probe changes in the αS conformation by single molecule Förster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes as well as their implications for hNatB substrate recognition and specific inhibition of the interaction with αS. Beyond the study of αS and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling.

The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacteria's ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key "SOS activation signal." Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that allow LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response.

Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNA Pyl pairs from Methanosarcina mazei ( Mm ) and Methanosarcina barkeri ( Mb ) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNA Pyl pair from Candidatus Methanomethylophilus alvus Mx1201 ( Ma ) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb / Mm PylRSs. An open question is how to alter Ma PylRS specificity to encode specific ncAAs with high efficiency. Prior work focused on "transplanting" ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm / Mb PylRSs in Ma PylRS. Here, we found that this strategy produced low-efficiency Ma PylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m -methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient Ma PylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 μM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All Ma PylRS variants identified through selection had at least two different active site residues when compared with their Mb PylRS counterparts. We conclude that Ma and Mm / Mb PylRSs are sufficiently different that "active site transplantation" yields suboptimal Ma GCE systems. This work establishes a paradigm for expanding the utility of the promising Ma PylRS/tRNA Pyl GCE platform.

We report an analytical bond energy from bond orders and populations (BEBOP) model that provides intramolecular bond energy decompositions for chemical insight into the thermochemistry of molecules. The implementation reported here employs a minimum basis set Mulliken population analysis on well-conditioned Hartree-Fock orbitals to decompose total electronic energies into physically interpretable contributions. The model's parametrization scheme is based on atom-specific parameters for hybridization and atom pair-specific parameters for short-range repulsion and extended Hückel-type bond energy term fitted to reproduce CBS-QB3 thermochemistry data. The current implementation is suitable for molecules involving H, Li, Be, B, C, N, O, and F atoms, and it can be used to analyze intramolecular bond energies of molecular structures at optimized stationary points found from other computational methods. This first-generation model brings the computational cost of a Hartree-Fock calculation using a large triple-ζ basis set, and its atomization energies are comparable to those from widely used hybrid Kohn-Sham density functional theory (DFT, as benchmarked to 109 species from the G2/97 test set and an additional 83 reference species). This model should be useful for the community by interpreting overall ab initio molecular energies in terms of physically insightful bond energy contributions, e.g., bond dissociation energies, resonance energies, molecular strain energies, and qualitative energetic contributions to the activation barrier in chemical reaction mechanisms. This work reports a critical benchmarking of this method as well as discussions of its strengths and weaknesses compared to hybrid DFT (i.e., B3LYP, M062X, PBE0, and APF methods), and other cost-effective approximate Hamiltonian semiempirical quantum methods (i.e., AM1, PM6, PM7, and DFTB3).

Arginylation is an understudied post-translational modification (PTM) involving the transfer of arginine to aspartate or glutamate sidechains in a protein. Among the targets of this PTM is α-synuclein (αS), a neuronal protein involved in regulating synaptic vesicles. The aggregation of αS is implicated in neurodegenerative diseases, particularly in Parkinson's disease, and arginylation has been found to protect against this pathological process. Arginylated αS has been studied through semisynthesis involving multipart native chemical ligation (NCL), but this can be very labor-intensive with low yields. Here, we present a facile way to introduce a mimic of the arginylation modification into a protein of interest, compatible with orthogonal installation of labels such as fluorophores. We synthesize bromoacetyl arginine and react it with recombinant, site-specific cysteine mutants of αS. We validate the mimic by testing the vesicle binding affinity of mimic-arginylated αS, as well as its aggregation kinetics and monomer incorporation into fibrils, and comparing these results to those of authentically arginylated αS produced through NCL. In cultured neurons, we compare the fibril seeding capabilities of preformed fibrils carrying a small percentage of arginylated αS. We find that, consistent with authentically arginylated αS, mimic-arginylated αS does not perturb the protein's native function but alters aggregation kinetics and monomer incorporation. Both mimic and authentically modified αS suppress aggregation in neuronal cells. Our results provide further insight into the neuroprotective effects of αS arginylation, and our alternative strategy to generate arginylated αS enables the study of this PTM in proteins not accessible through NCL.

A variety of post-translational modifications (PTMs) are believed to regulate the behavior and function of α-synuclein (αS), an intrinsically disordered protein that mediates synaptic vesicle trafficking. Fibrils of αS are implicated in neurodegenerative disorders such as Parkinson's disease. In this study, we used chemical synthesis and biophysical techniques to characterize the neuroprotective effects of glutamate arginylation, a hitherto little characterized PTM in αS. We developed semisynthetic routes combining peptide synthesis, unnatural amino acid mutagenesis, and native chemical ligation (NCL) to site-specifically introduce the PTM of interest along with fluorescent probes into αS. We synthesized the arginylated glutamate as a protected amino acid, as well as a novel ligation handle for NCL, in order to generate full-length αS modified at various individual sites or a combination of sites. We assayed the lipid-vesicle binding affinities of arginylated αS using fluorescence correlation spectroscopy (FCS) and found that arginylated αS has the same vesicle affinity compared to control protein, suggesting that this PTM does not alter the native function of αS. On the other hand, we studied the aggregation kinetics of modified αS and found that arginylation at E83, but not E46, slows aggregation and decreases the percentage incorporation of monomer into fibrils in a dose-dependent manner. Arginylation at both sites also resulted in deceleration of fibril formation. Our study represents the first synthetic strategy for incorporating glutamate arginylation into proteins and provides insight into the neuroprotective effect of this unusual PTM.

Thioamide-containing amino acids have been shown to quench a wide range of fluorophores through distinct mechanisms. Here, we quantitatively analyze the mechanism through which the thioamide functional group quenches the fluorescence of p -cyanophenylalanine (Cnf), tyrosine (Tyr), and tryptophan (Trp). By comparing PyRosetta simulations to published experiments performed on polyproline ruler peptides, we corroborate previous findings that both Cnf and Tyr quenching occurs via Förster resonance energy transfer (FRET), while Trp quenching occurs through an alternate mechanism such as Dexter transfer. Additionally, optimization of the peptide sampling scheme and comparison of thioamides attached to the peptide backbone and side chain revealed that the significant conformational restriction associated with the thioamide moiety results in a high sensitivity of the apparent FRET efficiency to underlying conformational differences. Moreover, by computing FRET efficiencies from structural models using a variety of approaches, we find that quantitative accuracy in the role of Coulomb coupling is required to explain contributions to the observed quenching efficiency from individual structures on a detailed level. Last, we demonstrate that these additional considerations improve our ability to predict thioamide quenching efficiencies observed during binding of thioamide-labeled peptides to fluorophore-labeled variants of calmodulin.

Thioamide substitutions of the peptide backbone have been shown to stabilize therapeutic and imaging peptides toward proteolysis. In order to rationally design thioamide modifications, we have developed a novel Rosetta custom score function to classify thioamide positional effects on proteolysis in substrates of serine and cysteine proteases. Peptides of interest were docked into proteases using the FlexPepDock application in Rosetta. Docked complexes were modified to contain thioamides parametrized through the creation of custom atom types in Rosetta based on ab intio simulations. Thioamide complexes were simulated, and the resultant structural complexes provided features for machine learning classification as the decomposed values of the Rosetta score function. An ensemble, majority voting model was developed to be a robust predictor of previously unpublished thioamide proteolysis holdout data. Theoretical control simulations with pseudo-atoms that modulate only one physical characteristic of the thioamide show differential effects on prediction accuracy by the optimized voting classification model. These pseudo-atom model simulations, as well as statistical analyses of the full thioamide simulations, implicate steric effects on peptide binding as being primarily responsible for thioamide positional effects on proteolytic resistance.

As recognition of the abundance and relevance of intrinsically disordered proteins (IDPs) continues to grow, demand increases for methods that can rapidly predict the conformational ensembles populated by these proteins. To date, IDP simulations have largely been dominated by molecular dynamics (MD) simulations, which require significant compute times and/or complex hardware. Recent developments in MD have afforded methods capable of simulating both ordered and disordered proteins, yet to date, accurate fold prediction from a sequence has been dominated by Monte Carlo (MC)-based methods such as Rosetta. To overcome the limitations of current approaches in IDP simulation using Rosetta while maintaining its utility for modeling folded domains, we developed PyRosetta-based algorithms that allow for the accurate de novo prediction of proteins across all degrees of foldedness along with structural ensembles of disordered proteins. Our simulations have accuracy comparable to state-of-the-art MD with vastly reduced computational demands.

The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to derepression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally perturbing probe of the E. coli RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength and demonstrate that a mobile loop encoding LexA residues 75-84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.

Thioamide substitutions in peptides can be used as fluorescence quenchers in protease sensors and as stabilizing modifications of hormone analogs. To guide these applications in the context of serine proteases, we here examine the cleavage of several model substrates, scanning a thioamide between the P3 and P3' positions, and identify perturbing positions for thioamide substitution. While all serine proteases tested were affected by P1 thioamidation, certain proteases were also significantly affected by other thioamide positions. We demonstrate how these findings can be applied by harnessing the combined P3/P1 effect of a single thioamide on kallikrein proteolysis to protect two key positions in a neuropeptide Y-based imaging probe, increasing its serum half-life to >24 h while maintaining potency for binding to Y1 receptor expressing cells. Such stabilized peptide probes could find application in imaging cell populations in animal models or even in clinical applications such as fluorescence-guided surgery.

Post-translational modifications (PTMs) impact the pathological aggregation of α-synuclein (αS), a hallmark of Parkinson's disease (PD). Here, we synthesize αS phosphorylated at tyrosine 39 (pY 39 ) through a novel route using in vitro enzymatic phosphorylation of a fragment followed by ligation to form the full-length protein. We can execute this synthesis in combination with unnatural amino acid mutagenesis to include two fluorescent labels for Förster resonance energy transfer (FRET) studies. We determine the effect of pY 39 on the aggregation of αS and compare our authentically phosphorylated material to the corresponding glutamate 39 "phosphomimetic." Intriguingly, we find that αS-pY 39 can either accelerate or decelerate aggregation, depending on the fraction of phosphorylated protein. The αS-E 39 mutant can qualitatively reproduce some, but not all, of these effects. FRET measurements and analysis of existing structures of αS help to provide an explanation for this phenomenon. Our results have important implications for the treatment of PD patients with tyrosine kinase inhibitors and highlight the importance of validating phosphomimetics through studies of authentic PTMs.

Thioamidation as a posttranslational modification is exceptionally rare, with only a few reported natural products and exactly one known protein example (methyl-coenzyme M reductase from methane-metabolizing archaea). Recently, there has been significant progress in elucidating the biosynthesis and function of several thioamide-containing natural compounds. Separate developments in the chemical installation of thioamides into peptides and proteins have enabled cell biology and biophysical studies to advance the current understanding of natural thioamides. This review highlights the various strategies used by Nature to install thioamides in peptidic scaffolds and the potential functions of this rare but important modification. We also discuss synthetic methods used for the site-selective incorporation of thioamides into polypeptides with a brief discussion of the physicochemical implications. This account will serve as a foundation for the further study of thioamides in natural products and their various applications.

Photoconvertible fluorophores can enable the visualization and tracking of a specific biomolecules, complexes, and cellular compartments with precise spatiotemporal control. The field of photoconvertible probes is dominated by fluorescent protein variants, which can introduce perturbations to the target biomolecules due to their large size. Here, we present a photoconvertible small molecule, termed CPX, that can be conjugated to any target through azide-alkyne cycloaddition ("click" reaction). To demonstrate its utility, we have applied CPX to study (1) trafficking of biologically relevant synthetic vesicles and (2) intracellular processes involved in transmission of α-synuclein (αS) pathology. Our results demonstrate that CPX can serve as a minimally perturbing probe for tracking the dynamics of biomolecules.

The fibrillary aggregation of the protein alpha synuclein (Asyn) is a hallmark of Parkinson's disease, and the identification of small molecule binding sites on fibrils is essential to the development of diagnostic imaging probes. A series of molecular modeling, photoaffinity labeling, mass spectrometry, and radioligand binding studies were conducted on Asyn fibrils. The results of these studies revealed the presence of three different binding sites within fibrillar Asyn capable of binding small molecules with moderate to high affinity. A knowledge of the amino acid residues in these binding sites will be important in the design of high affinity probes capable of imaging fibrillary species of Asyn.

Improvements in genetic code expansion have made preparing proteins with diverse functional groups almost routine. Nonetheless, unnatural amino acids (Uaas) pose theoretical burdens on protein solubility, and determinants of position-specific tolerability to Uaas remain underexplored. To broadly examine associations, we systematically assessed the effect of substituting the fluorescent Uaa, acridonylalanine, at more than 50 chemically, evolutionarily, and structurally diverse residues in two bacterial proteins: LexA and RecA. Surprisingly, properties that ostensibly contribute to Uaa tolerability-such as conservation, hydrophobicity, or accessibility-demonstrated no consistent correlations with resulting protein solubility. Instead, solubility is closely dependent on the location of the substitution within the overall tertiary structure, suggesting that intrinsic properties of protein domains, and not individual positions, are stronger determinants of Uaa tolerability. Consequently, those who seek to install Uaas in new target proteins should consider broadening, rather than narrowing, the types of residues screened for Uaa incorporation.

Small-molecule fluorescent probes are powerful tools for chemical biology; however, despite the large number of probes available, there is still a need for a simple fluorogenic scaffold, which allows for the rational design of molecules with predictable photophysical properties and is amenable to concise synthesis for high-throughput screening. Here, we introduce a highly modular quinoline-based probe containing three strategic domains that can be easily engineered and optimized for various applications. Such domains are allotted for (1) compound polarization, (2) tuning of photophysical properties, and (3) structural diversity. We successfully synthesized our probes in two steps from commercially available starting materials in overall yields of up to 95%. Facile probe synthesis was permitted by regioselective palladium-catalyzed cross-coupling, which enables combinatorial development of structurally diverse quinoline-based fluorophores. We have further applied our probes to live-cell imaging, utilizing their unique two-stage fluorescence response to intracellular pH. These studies provide a full demonstration of our strategy in rational design and stream-lined probe discovery to reveal the diverse potential of quinoline-based fluorescent compounds.

Peptide hormones are attractive as injectable therapeutics and imaging agents, but they often require extensive modification by mutagenesis and/or chemical synthesis to prevent rapid in vivo degradation. Alternatively, the single-atom, O-to-S modification of peptide backbone thioamidation has the potential to selectively perturb interactions with proteases while preserving interactions with other proteins, such as target receptors. Here, we use the validated diabetes therapeutic, glucagon-like peptide-1 (GLP-1), and the target of clinical investigation, gastric inhibitory polypeptide (GIP), as proof-of-principle peptides to demonstrate the value of thioamide substitution. In GLP-1 and GIP, a single thioamide near the scissile bond renders these peptides up to 750-fold more stable than the corresponding oxopeptides toward cleavage by dipeptidyl peptidase 4, the principal regulator of their in vivo stability. These stabilized analogues are nearly equipotent with their parent peptide in cyclic AMP activation assays, but the GLP-1 thiopeptides have much lower β-arrestin potency, making them novel agonists with altered signaling bias. Initial tests show that a thioamide GLP-1 analogue is biologically active in rats, with an in vivo potency for glycemic control surpassing that of native GLP-1. Taken together, these experiments demonstrate the potential for thioamides to modulate specific protein interactions to increase proteolytic stability or tune activation of different signaling pathways.

Accurate imaging of nanometer-sized structures and morphologies is essential to characterizing amyloid species formed at various stages of amyloid aggregation. In this article, we examine the effect of different drying procedures on the final morphology of surface-mediated fibrils formed during the incubation period, which may then be mistaken as oligomers or protofibrils intentionally formed in solution for a particular study. Atomic force microscopy results show that some artifacts, such as globules, flakelike structures, and even micrometer-long fibrils, can be produced under various drying conditions. We also demonstrate that one can prevent drying artifacts by using an appropriate spin-coating procedure to dry amyloid samples. This procedure can bypass the wetting/dewetting transition of the liquid layer during the drying process and preserve the structure of interest on the substrate without generating drying artifacts.

Fibrillar aggregates of the protein α-synuclein (αS) are one of the hallmarks of Parkinson's disease. Here, we show that measuring the fluorescence polarization (FP) of labels at several sites on αS allows one to monitor changes in the local dynamics of the protein after binding to micelles or vesicles, and during fibril formation. Most significantly, these site-specific FP measurements provide insight into structural remodeling of αS fibrils by small molecules and have the potential for use in moderate-throughput screens to identify small molecules that could be used to treat Parkinson's disease.

The expression of proteins containing unnatural amino acids through suppression of a stop codon can be limited by truncation due to competition with release factors. When the site of incorporation is near the C-terminus, it may not be feasible to separate the full-length unnatural amino acid protein from the truncated form. We report a simple, traceless procedure that allows one to isolate the desired protein using a C-terminal intein fusion.

Here we present a label-free method for studying the mechanism of surface effects on amyloid aggregation. In this method, spin-coating is used to rapidly dry samples, in a homogeneous manner, after various incubation times. This technique allows the control of important parameters for self-assembly, such as the surface concentration. Atomic force microscopy is then used to obtain high-resolution images of the morphology. While imaging under dry conditions, we show that the morphologies of self-assembled aggregates of a model amyloid-β peptide, Aβ(12-28), are strongly influenced by the local surface concentration. On mica surfaces, where the peptides can freely diffuse, homogeneous, self-assembled protofibrils formed spontaneously and grew longer with longer subsequent incubation. The surface fibrillization rate was much faster than the rates of fibril formation observed in solution, with initiation occurring at much lower concentrations. These data suggest an alternative pathway for amyloid formation on surfaces where the nucleation stage is either bypassed entirely or too fast to measure. This simple preparation procedure for high-resolution atomic force microscopy imaging of amyloid oligomers and protofibrils should be applicable to any amyloidogenic protein species.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25-95 μM) and low affinities (Kd = 400-700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B-C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1'-myoinositol-4',5'-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

Thioamide quenchers can be paired with compact fluorophores to design "turn-on" fluorescent protease substrates. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases, including trypsin, chymotrypsin, pepsin, thermolysin, papain, and calpain. Since thioamides quench some fluorophores red-shifted from those naturally occurring in proteins, this technique can be used for real time monitoring of protease activity in crude preparations of virtually any protease. We demonstrate the value of this method in three model applications: (1) characterization of papain enzyme kinetics using rapid-mixing experiments, (2) selective monitoring of cleavage at a single site in a peptide with multiple proteolytic sites, and (3) analysis of the specificity of an inhibitor of calpain in cell lysates.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in Escherichia coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here, we report the synthesis of Acd in 87% yield over five steps from Tyr and the identification of an Acd synthetase by screening candidate enzymes previously evolved from Methanococcus janaschii Tyr synthetase for unnatural amino acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with select natural amino acids and Förster resonance energy transfer (FRET) interactions with common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu(3+) to monitor protein folding and binding interactions.

Previously we have shown that thioamides can be incorporated into proteins as minimally perturbing fluorescence-quenching probes to study protein dynamics, folding, and aggregation. Here, we show that the spontaneity of photoinduced electron transfer between a thioamide and an excited fluorophore is governed by the redox potentials of each moiety according to a Rehm-Weller-type model. We have used this model to predict thioamide quenching of various common fluorophores, and we rigorously tested more than a dozen examples. In each case, we found excellent agreement between our theoretical predictions and experimental observations. In this way, we have been able to expand the scope of fluorophores quenched by thioamides to include dyes suitable for microscopy and single-molecule studies, including fluorescein, Alexa Fluor 488, BODIPY FL, and rhodamine 6G. We describe the photochemistry of these systems and explore applications that demonstrate the utility of thioamide quenching of fluorescein to studying protein folding and proteolysis.

We have recently shown that p-cyanophenylalanine (Cnf) and a thioamide can be used as a minimally perturbing Förster resonant energy transfer (FRET) pair to monitor protein conformation. We have also shown that thioamide analogues of natural amino acids can be incorporated into full-sized proteins through native chemical ligation. For intermolecular studies with Cnf/thioamide FRET pairs, Cnf can be incorporated into proteins expressed in Escherichia coli through unnatural amino acid mutagenesis using a Cnf-specific tRNA synthetase. For intramolecular studies, a Cnf-labeled protein fragment can be expressed in E. coli and then ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for rapid access to double-labeled proteins with a minimum of unnecessary chemical synthesis. We demonstrate the utility of this approach by studying the binding of peptides to the protein calmodulin and by determining the orientation of the N- and C-termini in the amyloidogenic protein α-synuclein.

Thioamide modifications of the peptide backbone are used to perturb secondary structure, to inhibit proteolysis, as photoswitches, and as spectroscopic labels. Thus far, their incorporation has been confined to single peptides synthesized on solid phase. We have generated thioamides in C-terminal thioesters or N-terminal Cys fragments and examined their compatibility with native chemical ligation conditions. Most sequence variants can be coupled in good yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations. Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track conformational changes during aggregation of labeled α-synuclein.

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond-a single atom substitution of the peptide backbone-can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetime measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a large number of measurements to model protein folding., (© 2012 American Chemical Society)

Methods for synthetically manipulating protein structure enable greater flexibility in the study of protein function. Previous characterization of the Escherichia coli aminoacyl tRNA transferase (AaT) has shown that it can modify the N-terminus of a protein with an amino acid from a tRNA or a synthetic oligonucleotide donor. Here, we demonstrate that AaT can efficiently use a minimal adenosine substrate, which can be synthesized in one to two steps from readily available starting materials. We have characterized the enzymatic activity of AaT with aminoacyl adenosyl donors and found that reaction products do not inhibit AaT. The use of adenosyl donors removes the substrate limitations imposed by the use of synthetases for tRNA charging and avoids the complex synthesis of an oligonucleotide donor. Thus, our AaT donors increase the potential substrate scope and reaction scale for N-terminal protein modification under conditions that maintain folding.

Decreasing the size of spectroscopic probes can afford higher-resolution structural information from fluorescence experiments. Therefore, we have developed p-cyanophenylalanine (Cnf) and backbone thioamides as a fluorophore/quencher pair. Through the examination of a series of thiopeptides, we have determined the working distance for this pair to be 8-30 Å. We have also carried out a proof-of-principle protein-folding experiment in which a Cnf/thioamide-labeled version of villin headpiece HP35 was thermally unfolded while the Cnf/thioamide distance was monitored by fluorescence. For a given protein, thioamide substitutions could be used to track motions with a much greater number of measurements than for current fluorescence probes, providing a dense array of data with which to model conformational changes.

A 28 residue "retro" beta-peptide has been designed to self-assemble as a tetramer, successfully recapitulating the octameric beta-peptide bundle complex Zwit-1F. Z28, the largest beta-peptide synthesized to date, was achieved in a linear, microwave-assisted synthesis in 19% isolated yield and high purity. Z28 forms the expected tetrameric complex and is more stable and cooperative in its folding than Zwit-1F. The successful design and synthesis of Z28 is an important step toward true beta-amino acid proteins.

Proteins composed of alpha-amino acids are essential components of the machinery required for life. Stanley Miller's renowned electric discharge experiment provided evidence that an environment of methane, ammonia, water, and hydrogen was sufficient to produce alpha-amino acids. This reaction also generated other potential protein building blocks such as the beta-amino acid beta-glycine (also known as beta-alanine); however, the potential of these species to form complex ordered structures that support functional roles has not been widely investigated. In this report we apply a variety of biophysical techniques, including circular dichroism, differential scanning calorimetry, analytical ultracentrifugation, NMR and X-ray crystallography, to characterize the oligomerization of two 12-mer beta3-peptides, Acid-1Y and Acid-1Y*. Like the previously reported beta3-peptide Zwit-1F, Acid-1Y and Acid-1Y* fold spontaneously into discrete, octameric quaternary structures that we refer to as beta-peptide bundles. Surprisingly, the Acid-1Y octamer is more stable than the analogous Zwit-1F octamer, in terms of both its thermodynamics and kinetics of unfolding. The structure of Acid-1Y, reported here to 2.3 A resolution, provides intriguing hypotheses for the increase in stability. To summarize, in this work we provide additional evidence that nonnatural beta-peptide oligomers can assemble into cooperatively folded structures with potential application in enzyme design, and as medical tools and nanomaterials. Furthermore, these studies suggest that nature's selection of alpha-amino acid precursors was not based solely on their ability to assemble into stable oligomeric structures.

Folded polymers in nature are assembled from simple monomers and adopt complex folded structures through networks of stabilizing noncovalent interactions. These interactions define secondary and tertiary structure and in most cases specify a unique three-dimensional architecture. Individual secondary or tertiary structures can also associate with one another to form multi-subunit quaternary structures. Nonnatural folded polymers have potential for similar structural versatility. Here we describe a pair of beta3-peptides whose sequences were designed to promote a 14-helix structure in water, favor hetero-oligomer formation, and disfavor nonspecific aggregation. These beta3-peptides assemble noncovalently into a well-defined hetero-oligomer characterized by a defined stoichiometry, a highly stabilized secondary structure, and a cooperative melting transition (TM > 55 degrees C). This work demonstrates that beta3-peptides can assemble into defined, cooperatively folded quaternary structures and constitutes an important step toward designing protein-like assemblies from nonnatural polymers.

The N -methyl-D-aspartate (NMDA) receptor plays a central role in learning and memory in the mammalian CNS. At normal neuronal resting membrane potentials, the pore of this glutamate-gated ion channel is blocked by a Mg(2+) ion. Previous work suggests that the Mg(2+) binding site is quite novel, involving several asparagine residues and a cation-pi interaction between Mg(2+) and a conserved tryptophan in the pore. Using unnatural amino acid mutagenesis, we show that no such cation-pi interaction exists. The implicated tryptophan instead appears to play a structural role that can only be fulfilled by a rigid, flat, hydrophobic residue. This is the first demonstration of unnatural amino acid incorporation in the NMDA receptor, and it opens the way for future investigations of this pivotal neuroreceptor.

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.

The binding of three distinct agonists-acetylcholine (ACh), nicotine, and epibatidine-to the nicotinic acetylcholine receptor has been probed using unnatural amino acid mutagenesis. ACh makes a cation-pi interaction with Trp alpha149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation-pi interaction and the backbone hydrogen bond. A simple structural model that considers only possible interactions with Trp alpha149 suggests that a novel aromatic C-H...O=C hydrogen bond further augments the binding of epibatidine. These studies illustrate the subtleties and complexities of the interactions between drugs and membrane receptors and establish a paradigm for obtaining detailed structural information.

A series of tethered quaternary ammonium derivatives of Tyr have been incorporated into the binding site of the nicotinic acetylcholine receptor (nAChR) using the in vivo nonsense suppression method, producing constitutively active (self-gating) receptors. We have incorporated primary, secondary, and tertiary amine tethered agonists to give receptors whose constitutive activity can be modulated by pH. Lowering the pH protonates the tethered amine, giving it a positive charge and allowing it to reversibly activate the receptor. Tertiary and secondary tethered amines, TyrO3T and TyrO3S, have been successfully incorporated at alpha149 in the nAChR. Constitutive currents at pH 5.5 are 6 times those at pH 9.0. The pKa of TyrO3T in the binding site appears to be 6 or lower, differing substantially from its pKa in solution ( approximately 9.3). This local pKa perturbation has substantial implications for pharmacological research on the nAChR: of the tertiary agonists considered, noracetylcholine experiences this pKa perturbation, while nicotine does not.