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The bacterium Staphylococcus aureus produces an array of anti-inflammatory molecules that prevent the innate immune system from recognizing it as a pathogen and clearing it from the host. In the acute phase of inflammation, our immune system relies on neutrophils to clear invading bacteria. Recently, novel classes of secreted proteins from S. aureus, including the Extracellular Adherence Protein (EAP) family (Stapels et al., Proc Natl Acad Sci USA 111:13187-13192, 2014) and the Staphylococcal Peroxidase Inhibitor (SPIN), (unpublished work) have been identified as highly selective inhibitors acting on Neutrophil Serine Proteases (NSPs) and myeloperoxidase (MPO) respectively. SPIN is a protein found only in Staphylococci, with no sequence homology to any known proteins. Solution NMR structural studies of SPIN are therefore expected to provide a deeper understanding of its interaction with MPO. In this study, we report the backbone and side-chain 1H, 15N, and 13C resonance assignments of SPIN. Furthermore, using the chemical shifts of these resonances, we predicted the secondary structure of SPIN in solution via the TALOS-N server. The assignment data has been deposited in the BMRB data bank under Accession No. 27069.

Previous research has indicated that long-chain fatty acids can bind myoglobin (Mb) in an oxygen-dependent manner. This suggests that oxy-Mb may play an important role in fuel delivery in Mb-rich muscle fibers (e.g. type I fibers and cardiomyocytes), and raises the possibility that Mb also serves as an acylcarnitine-binding protein. We report for the first time the putative interaction and affinity characteristics for different chain lengths of both fatty acids and acylcarnitines with oxy-Mb using molecular dynamic simulations and isothermal titration calorimetry experiments. We found that short- to medium-chain fatty acids or acylcarnitines (ranging from C2:0 to C10:0) fail to achieve a stable conformation with oxy-Mb. Furthermore, our results indicate that C12:0 is the minimum chain length essential for stable binding of either fatty acids or acylcarnitines with oxy-Mb. Importantly, the empirical lipid binding studies were consistent with structural modeling. These results reveal that: (i) the lipid binding affinity for oxy-Mb increases as the chain length increases (i.e. C12:0 to C18:1), (ii) the binding affinities of acylcarnitines are higher when compared with their respective fatty acid counterparts, and (iii) both fatty acids and acylcarnitines bind to oxy-Mb in 1:1 stoichiometry. Taken together, our results support a model in which oxy-Mb is a novel regulator of long-chain acylcarnitine and fatty acid pools in Mb-rich tissues. This has important implications for physiological fuel management during exercise, and relevance to pathophysiological conditions (e.g. fatty acid oxidation disorders and cardiac ischemia) where long-chain acylcarnitine accumulation is evident.

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Forster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post- and cotranslational signaling processes.

Protein beverages have been in demand due to an increasing consumers’ interest in healthy eating habit. However, there is an increased concern on the use of genetic modified (GM) ingredient in the food product. This study aimed to develop protein hydrolysate beverages using a non-GM soybean (R08-4004/high protein line) grown in Arkansas. Protein isolate was prepared from the soybean using alkaline method (pH 9.5). Due to its poor solubility in acidic condition, alcalase 2.4 L (food grade protease) hydrolyzed soy protein was used to develop a beverage containing 20 g protein per serving (500 mL). Three flavored beverages: Chai tea (C), tangerine (T), and mixed berries (MB) were prepared using bitter blocker, masking agent, and citric acid to minimize an unpleasant bitter taste developed in the soy hydrolysates. Protein solubility, pH, microbial growth, instrumental color parameters, and turbidity were measured to evaluate the shelf-life stability of the beverages at refrigerated storage (5 °C) for 42 days. Beverages T and MB received overall highest scores from the sensory panel. Citric acid alone or in combination with bitter blocker or masking agent lowered the bitterness. Pasteurization (90–95 °C for 5 min) was effective in preventing microbial growth. Although pH remained constant, decrease in protein solubility and color changes were observed over the storage time in all the three flavored beverages. Cloudiness in beverage C increased over the storage period while beverages T and MB were very stable. Overall, T and MB flavored beverages have the potential for commercial application.

Multidimensional nuclear magnetic resonance spectroscopy (NMR) is a very versatile technique which is routinely used for the characterization of 3D structure, backbone dynamics, and intermolecular interactions of proteins in solution at atomic resolution. NMR spectroscopy as an analytical technique has come of age. Availability of higher field spectrometers, equipped with cryoprobes, has drastically increased the sensitivity and resolution of NMR data. In recent years, several new pulse sequences have been developed which have significantly diminished the perceived molecular-size limitation of NMR spectroscopy. Currently, determination of 3D solution structures of proteins, with molecular mass in the 100 kDa range, is no longer impossible. Like X-ray crystallography and electron microscopy, multidimensional NMR spectroscopy has till recently been only conceived as an in vitro technique which can help in the structural characterization of proteins. However, several interesting questions pertaining to the physiological relevance of the protein structures determined using these in vitro techniques have been raised. For example, are the 3D structures of isolated proteins determined under in vitro conditions different than when they are present in a crowded cellular environment with other proteins and biomolecules? Do protein-ligand structural interactions characterized under in vitro conditions bear resemblance to those which occur in the cellular milieu? Most importantly, do post-translational modifications which occur during protein maturation cause significant change in the conformation of proteins? These intriguing questions can be satisfactorily addressed by the recently developed in-cell NMR spectroscopy. In-cell NMR spectroscopy is an emerging bioanalytical technique which can be employed to obtain atomic-level information on the structure, folding, dynamics, and interactions of proteins in a cell. Two-dimensional correlation experiments involving NMR sensitive atomic nuclei (such as, 1H, 13C and 15N) are primary to the characterization of protein structure and their interactions. 1H-15N/1H-13C HSQC (Heteronuclear Single Quantum Coherence) is the most informative and widely employed 2D correlation experiment used in in-cell NMR spectroscopy. 1H-15N HSQC is preferred over the 1H-13C HSQC experiment due to its simplicity and significantly lower costs of preparation of 15N-enriched protein samples. Each 1H-15N correlated peak in a 1H-15N HSQC spectrum represents an amino acid in a particular backbone conformation of a protein. Therefore, 1H-15N HSQC spectrum of a protein can be expected to be unique (Figure 1). Although the total number of 1H-15N resonance crosspeaks in a given HSQC spectrum remains the same, local changes in chemical microenvironment of the NMR sensitive atomic nuclei (1H and 15N), effected either due to ligand binding or due to conformational changes induced in a protein, causes difference(s) in the chemical shift value of definite atomic nuclei. Differences in the chemical shift values of the participating nuclei, measured as a composite chemical shift value [Δδcomposite=Δδ1H+Δ15N/13C], provide valuable information on specific amino acid residues involved in ligand binding or in a conformational change. This rationale applies to both in vitro and in vivo experiments. Figure 1 1H-15N HSQC spectra of human fibroblast growth factor-1 in the absence (red) and presence (blue) of heparin analogue. There are unlimited applications for in-cell NMR spectroscopy. Three-dimensional structures of proteins can be determined in a cellular environment and these 3D structures would bear more relevance than those which are solved under in vitro conditions. Similarly, protein structures are not plastic because proteins exhibit interesting backbone as well as side-chain dynamics in the picoseconds to micro/millisecond time scale. In-cell NMR is an apt technique to monitor these dynamic protein motions. In addition, proteins are targets for most of the pharmaceutical drugs and the inherent capability of in-cell NMR to characterize molecular interactions at atomic resolution is immensely valuable towards efforts to design novel, specific, and potent therapeutic principles against multitude of diseases. Further, protein aggregation is currently plaguing pharmaceutical industries and billions of dollars are lost due to the problem of formation of inclusion bodies (protein aggregates) during overexpression of recombinant proteins in heterologous hosts. In-cell NMR spectroscopy provides a unique avenue to study in vivo molecular mechanisms that govern the partition between productive folding of proteins to their biologically active conformation and unproductive pathways leading to aggregated state(s). Post-translational modifications of proteins either serve as cellular regulatory mechanism(s) that critically alters protein-protein interactions or they facilitate the specific targeting of proteins in to different sub-cellular compartments. In-cell NMR spectroscopy is an invaluable technique to trace the different steps of protein maturation and understand how they shape the folding and function of proteins. Lastly, in recent years, there has been increased interest in the characterization of intrinsically disordered proteins (IDPs). This class of proteins lack elaborate structure(s) but serve as hub(s) for protein-protein interactions that regulate key cellular processes. Recently, in-cell NMR spectroscopy has been successfully employed to track the structure and folding of IDPs in a cellular environment. In conclusion, the scope of applications of in-cell NMR seems limitless and future development(s) in the NMR spectrometer hardware, pulse sequences and methods of overexpression of isotope enriched proteins will help unravel the full potential of this fascinating technique.

The mechanisms of nonclassical export of signal peptide-less proteins remain insufficiently understood. Here we demonstrate that stress-induced unconventional export of FGF1, a potent and ubiquitously expressed mitogenic and proangiogenic protein, is associated with and dependent on the formation of membrane blebs and localized cell surface exposure of phosphatidylserine. In addition, we found that the differentiation of promonocytic cells results in massive FGF1 release, which also correlates with membrane blebbing and exposure of phosphatidylserine. These findings indicate that the externalization of acidic phospholipids could be used as a pharmacological target to regulate the availability of FGF1 in the organism.

Fibroblast Growth Factor (FGF) signaling plays a vital role in a wide range of cellular responses (Detillieux et al., 2003; Freeman et al., 2003). The activation of FGF Receptor (FGFR) is a crucial step in the diverse FGF signaling pathway ( Ayari & Soussi-Yanicostas, 2007). Various intraand extracellular modulators are involved in the FGF signaling pathway. Anosmin-1, an extracellular matrix associated glycosylated protein, is a newly identified modulator of the FGF-mediated signaling process (Bribian et al., 2006; GonzalezMartinez et al., 2004). Anosmin-1 is the product of KAL1 gene. The Kallman syndrome (KS) is a manifestation of the different loss-of-function mutations in the KAL1 gene. The most characteristic features of the Kallman syndrome are anosmia (lack of smell) and hypogonadotrophic hypogonadism (Maestre, 1856; Kallmann et al., 1944). The biological role(s) of Anosmin-1 has been studied both at the cellular and at the biochemical level. Anosmin-1 is suggested to be involved in cell adhesion, cell migration, cell proliferation as well as cell differentiation (Andrenacci et al., 2004; Andrenacci et al., 2006; Ardouin et al., 2000; Bribian et al., 2006; Cariboni et al., 2004; Ernest et al., 2007; Hardelin et al., 1999; Hu et al., 2004; Okubo et al., 2006; Robertson et al., 2001; Rugrali et al., 1996; Soussi-Yanicostas, 1996; Soussi-Yanicostas et al., 1998; Soussi-Yanicostas et al., 2002; Yanicostas et al., 2008;). Anosmin-1 is shown to be a heparin binding protein and formation of the heparin-Anosmin binary complex is believed to be crucial for the function of the protein (Bulow et al., 2002). Interestingly, recent reports suggest a direct interaction between the Nterminus of Anosmin-1 and the FGF-2/FGFR-1/HS ternary complex (Hu et al., 2009; Hu & Bouloux, 2010). The structure of Anosmin-1(Figure 1) comprises of an N-terminal cysteine-rich domain (CR), whey acidic like-protein domain (a-WAP), four fibronectin type III (FnIII) repeats and C-terminal histidine rich region. Recent reports suggest direct interactions of some of the structural domains of Anosmin-1, including a-WAP, with the fibroblast growth factor receptor(s). Although the Anosmin-FGFR binding interface is still not mapped, it is strongly believed that the interactions of Anosmin-1 with FGFR(s) regulates the activation of the receptor.

This is a response to a letter by Falk and Sinning (1) We recently identified the ankyrin region of cpSRP43 as the primary domain responsible for binding Alb3-Cterm during light-harvesting chlorophyll-binding protein (LHCP) targeting, an interaction shown to facilitate cpSRP43-dependent stimulation of cpSRP GTPases by Alb3-Cterm (2). Falk et al. (3), using only protein interaction assays, report that CD2CD3 of cpSRP43 forms the Alb3-Cterm binding interface, which appears inconsistent with the fact that CD3 is not required for LHCP integration (4) and that CD2 is not required for LHCP integration by a cpSRP54/cpFtsY-independent pathway that relies on cpSRP43/Alb3 (5). We suggested that buffer choice, including the use of glycerol, may play a role in why Falk et al. (3) observed μm rather than nm affinity for cpSRP43 constructs (2). The use of high concentrations of glycerol in isothermal titration calorimetry (ITC) is known to cause experimental artifacts (6). Our control experiments clearly show that the use of glycerol, even at 5% v/v, causes significant background heat changes (Fig. 1). In their letter, Sinning and Falk report a 13 μm affinity even in the absence of glycerol, suggesting glycerol may not be the primary cause for the reported differences. Although species-specific differences in Alb3-Cterm could explain the observed affinity differences, comparing GTPase stimulation by Arabidopsis and Pisum sativum Alb3-Cterm does not support this possibility (Fig. 2). FIGURE 1. Isothermal titration calorimetry investigation of the influence of glycerol in various buffers in buffer to buffer experiments. ITC was conducted by injecting a specific buffer/glycerol formulation into a sample well containing the same buffer/glycerol ... FIGURE 2. Comparison of the ability of Pisum sativum and Arabidopsis thaliana Alb3-Cterm peptide to stimulate cpSRP43-dependent GTP hydrolysis by the cpSRP GTPases. The effect of Alb3-Cterm on the GTP hydrolysis activity of cpSRP54 and cpFtsY was examined in the ... Published reports (2, 4, 5) supporting the physiological relevance of high affinity protein interactions still suggest that the low affinity of cpSRP43 for Alb3-Cterm reported by Falk et al. (3) stems from assay conditions unfavorable for observing the primary targeting interaction that takes place between Alb3-Cterm and the Ank region of cpSRP43. Importantly, buffers used by Falk et al. (3) in ITC and size-exclusion experiments do not support LHCP integration (Fig. 3). In addition, Ank-containing cpSRP43 constructs, including those that lack CD2 and/or CD3, are able to prevent binding of radiolabeled cpSRP43 to Alb3 in salt-washed thylakoids whereas chromodomains do not (Fig. 4). FIGURE 3. Buffer influence on LHCP integration. Salt-washed thylakoids in IBM were incubated with 5 mm ATP, 1 mm GTP, 12.5 μl of radiolabeled pLHCP translation product, and recombinant cpSRP43, cpSRP54, and cpFtsY (2). The final volume was brought to 150 ... FIGURE 4. Competition for cpSRP43 binding to the C terminus of Alb3 in salt-washed thylakoids. Salt-washed thylakoids (equivalent to 75 μg of chlorophyll) and 8 nmol of recombinant cpSRP43 construct as indicated were incubated for 15 min at 25 °C ...

A new strategy to prevent degradation of recombinant proteins caused by non-specific cleavage by thrombin is described. We demonstrate that degradation due to non-specific cleavage of recombinant protein mediated by thrombin can be completely prevented by separation of thrombin from the recombinant protein on spin columns packed with heparin-sepharose. This method is generally applicable to all recombinant proteins that require the thrombin for the cleavage of affinity tags for purification. To our knowledge, this is the first report of an efficient and reliable method for the separation of residual thrombin from purified recombinant proteins.

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.

Fibroblast growth factor (FGF)1 is released from cells as a constituent of a complex that contains the small calcium binding protein S100A13, and the p40 kDa form of synaptotagmin (Syt)1, through an ER-Golgi-independent stress-induced pathway. FGF1 and the other components of its secretory complex are signal peptide-less proteins. We examined their capability to interact with lipid bilayers by studying protein-induced carboxyfluorescein release from liposomes of different phospholipid (pL) compositions. FGF1, p40 Syt1, and S100A13 induced destabilization of liposomes composed of acidic but not of zwitterionic pL. We produced mutants of FGF1 and p40 Syt1, in which specific basic amino acid residues in the regions that bind acidic pL were substituted. The ability of these mutants to induce liposomes destabilization was strongly attenuated, and they exhibited drastically diminished spontaneous and stress-induced release. Apparently, the non-classical release of FGF1 and p40 Syt1 involves destabilization of membranes containing acidic pL.

Cardiotoxin analogue III (CTX III) is a low-molecular-mass all-α-sheet protein isolated from the Taiwan cobra (Naja naja atra) venom. A stable partially structured state similar to the ‘molten globule’ state has been identified for CTX III in a 3% (w/v) solution of 2,2,2-trichloroacetic acid at 298 K. This stable state has been structurally characterized using a variety of techniques such as CD, 1-anilinonaphthalene-8-sulphonate fluorescence binding, Fourier transform IR and two-dimensional NMR spectroscopy techniques. Direct assignment of the homonuclear two-dimensional NMR spectra of the protein in 3% trichloroacetic acid showed that drastic structural perturbation had not taken place in the protein and that the ‘intermediate’ state retained a significant portion of the native secondary-structural interactions. It is found that about 65% of the native α-sheet structural contacts are maintained in the partially structured state of CTX III in 3% trichloroacetic acid.

Human fibroblast growth factor (hFGF-1) is a approximately 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu(2+)) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu(2+) and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu(2+) appears to stabilize the protein bound to pS vesicles. Cu(2+) and lipid binding interface mapped using 2D (1)H-(15)N heteronuclear single quantum coherence experiments reveal that residues in beta-strand I contributes to the unique Cu(2+) binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and beta-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu(2+), additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu(2+)-induced non-classical secretion of hFGF-1.

S100A13 is a calcium binding chaperone protein, known to be involved in the non-classical export of signal peptide less proteins such as fibroblast growth factor (FGF-1) and interleukin-1α across the cell membrane. It has also been shown that the interaction of S100A13 with Annexin II, which exhibits an inducible flip-flop mechanism across the cell bilayer, helps the multiprotein release complex to traverse the membrane bilayer. The interaction of S100A13 and annexin II has been characterized using various biophysical techniques including multidimensional NMR spectroscopy. Results of the Isothermal titration calorimetry (ITC) experiments show that holo-S100A13 exhibits preferential binding to annexin II with high affinity in the micro molar rang compared to apo-S100A13. Equilibrium guanidine hydrochloride denaturation monitored by steady-state fluorescence and limited trypsin digestion analysis reveals holo-S100A13 is significantly stabilized upon binding to annexin II peptide compared to apo-S100A13. ANS (8-anilino-1-napthalene sulfonate) binding experiments indicates that the presence of annexin II peptide does not increase the solvent availability of hydrophobic residues in holo-S100A13. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 does not facilitate its interaction with the annexin II peptide, which is unique characteristic of S100A13. 1H-15N- HSQC NMR experiments reveal that the binding site of annexin II peptide on holo-S100A13 is modestly different from other S100/Annexin interactions. S100 proteins typically interact with more than one Annexin protein. In order to define the specificity of S100A13 for Annexin 2, the interaction of S100A13 with other Annexin peptides was characterized and data characterizing these interactions will be presented. Finally, defining the interaction between S100A13 and Annexin 2 peptide will give much needed insight into the non-classical release of the signal peptide-less protein Fibroblast Growth Factor-1

Human fibroblast growth factor (FGF) is an important protein that plays a role in morphogenesis, angiogenesis and wound healing. Because of these capabilities this protein is of medical interest. Unfortunately the lack of stability of FGF poses a problem to further research. Understanding the structure of FGF at the atomic level is necessary for designing novel FGF variants with enhanced stability and wound healing properties. The specific aim of this study is to test and record the structural stability of FGF through a gradient of salt concentrations. Through thermal denaturation, it was shown that the stability increased slightly with the increased salt concentration. The structure of hFGF-1 has been investigated using a variety of biophysical techniques such as trypsin digestion and circular dichroism. To ensure that the structure was maintained, N-15 enriched human fibroblast growth factor-1 was over expressed and purified. This sample will be used to test the structural stability of hFGF-1 by Heteronuclear Single Quantum Coherence (HSQC) using NMR spectroscopy. The hope of this study is to identify key properties that will allow the optimization of FGF stability. These data can then be used to develop novel mutants with enhanced stability and wound healing properties.

Fibroblast Growth Factor-1 (FGF-1) is a 16kDa heparin binding protein, which has been associated with a variety of important functions including angiogenesis and wound repair. In order for FGF-1 to enter the cell it must interact with the FGF-1 receptor on the cell surface. One vital residue involved in the binding of FGF-1 to the receptor is tryptophan 121 (W121). This study aims to examine the role of W121 on the conformation and functionality of FGF-1. Site-directed mutagenesis will be used to incorporate mutations at position 121. The effect of these mutations will be characterized using various biophysical techniques including fluorescence, CD, ITC, and mutli-dimesional NMR spectroscopy. As FGFs are involved in many crucial cellular processes, the gain from this study is expected to provide useful information on the regulation of the FGF signaling process.

Fibroblast growth factors (FGFs) are ∼ 16 kDa heparin binding proteins that regulate key cellular processes such as angiogenesis, differentiation, morphogenesis, wound healing and tumor growth. FGFRs consist of three extracellular ligand binding domains (D1, D2, D3), a single transmembrane helix, and cytoplasmic tyrosine kinase domain. Cell surface-bound HSPGs (heparan sulfate proteoglycans) supported dimerization or polymerization of the FGFRs are thought to be required to activate the signaling pathway. The D2 domain is suggested to bind with both HSPGs and FGFs to form a ternary complex. Xray and NMR solution structures of the D2 domain have been analyzed using the CAPTURE cation-pi program. The CAPTURE program indicates cation-pi interactions between residues Y10:R7 (Xray), W46:R58 (NMR) and possibly F92:K6 (Xray). Biophysical characterization of the mutants at each cation and pi pair, identified by CAPTURE, shows a significant destabilization resulting from the Y10A, W46A and R58E mutations. Results from differential scanning calorimetry show a reduction in melting temperature by 10-14 °C for Y10A, W46 and R58 mutants of D2. The reduction in the stability of the D2 domain is corroborated by results of ANS binding, thermal denaturation and a limited trypsin digestion experiments. The HSQC of D2 Y10A shows limited chemical shift perturbation of residues in the vicinity of the mutation site. The W46A and R58E mutations show significant 1H-15N chemical shift perturbations in their HSQC spectra. The results obtained in this study show that cation-pi interactions contribute significantly to the thermodynamic stability of proteins. In addition, our results indicate that cation-pi predictions made on solution NMR structures are more reliable than those predicted based on crystal structures.

S100A13 is a calcium binding chaperone protein that is known to be involved in the non-classical export of signal peptide-less proteins, such as fibroblast growth factor (FGF-1) and interleukin-1α,. It has also been shown that the interaction of S100A13 with Annexin II, which exhibits an inducible flip-flop mechanism across the cell bilayer, helps the multiprotein release complex to traverse the membrane bilayer. The interaction of S100A13 and Annexin 2 has been characterized using various biophysical techniques including Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), and multidimensional NMR spectroscopy. Results of the Isothermal titration calorimetry (ITC) experiments show that holo-S100A13 exhibits preferential binding to Annexin II with high affinity in the low micro molar range compared to apo-S100A13. Equilibrium guanidine hydrochloride denaturation monitored by steady-state fluorescence and limited trypsin digestion analysis both reveal holo-S100A13 to be stabilized upon binding with the Annexin II peptide. ANS (8-anilino-1-napthalene sulfonate) binding experiments indicate that the presence of Annexin II peptide does not increase the solvent accessiblity of hydrophobic residues in holo-S100A13, which is a unique characteristic of S100A13. 1H-15N- HSQC NMR experiments reveal the binding site of the Annexin II peptide on holo-S100A13 to be distinctly different from other S100/Annexin interactions. In order to define the specificity of S100A13 for Annexin II specifically, the interaction of S100A13 with other Annexin peptides was characterized by ITC. The information gained from this study provides valuable information on the interaction between S100A13 and the Annexin II peptide and gives much needed insight into the mysterious pathway of non-classical release of the signal peptide-less proteins.

Chloroplasts use chloroplast signal recognition particle (cpSRP) pathway to import important cargo like light harvesting chlorophyll protein (LHCP). cpSRP is unique among SRPs in being devoid of RNA. cpSRP consists of an evolutionarily conserved 54-kDa subunit (cpSRP54) and an unique 43-kDa subunit (cpSRP43). cpSRP43 subunit has four-ankyrin repeat domain at the N terminus and a C-terminal chromo domain (CD). The C-terminal CD of cpSRP43 has been shown to provide interaction sites for the cpSRP54 subunit. In addition, the chromodomain in the cpSRP43 subunit is also believed to be important for the formation of the transit complex with LHCP. Also, recent work on cpSRP 43 protein has shown that it mimics the shape and charge distribution of SRP RNA, that's missing from this system. In this context, we embarked on the structural characterization of the C-terminal CD using a variety of biophysical techniques including multidimensional NMR spectroscopy. Far UV circular dichroism spectrum of CD shows that the backbone of the protein is predominantly in the helical conformation. 1H-15N HSQC spectrum of CD is well- dispersed suggesting that the protein is structured. Complete resonance assignments (1H, 15N and 13C) in CD have been accomplished using a variety of triple resonance experiments. Chemical shift index plots show that CD is an alpha + beta protein. A detailed analysis of the three-dimensional solution structure of CD and its interaction with the cpSRP 54 peptide will be presented. The three-dimensional solution structure of CD provides valuable insights into the molecular mechanism underlying the post-translational transport and integration of LHCP on the thylakoid membrane.

Protein refolding is known to be dependent on factors such as protein concentration, temperature, pH, denaturant concentration, and several other factors. Protein folding and aggregation are competing reactions and the factors responsible for the partition between these two reactions are still not well understood. We have shown that lysozyme even at very high concentrations (10 mg/mL) is capable of refolding to near 100% activity when its disulfide bonds are intact. However, in the presence of a reducing agent such as DTT, lysozyme will aggregate significantly at very low concentrations (50 ug/mL). Furthermore, when refolding, denatured/reduced lysozyme is unable to refold in the absence of a redox mixture of reduced and oxidized glutathione. Previous studies conducted by Trivedi et. al have also demonstrated that the renaturation yield of lysozyme increases when co-refolding in the presence of other denatured states of basic proteins. We hypothesize that the renaturation yield of lysozyme will also increase when refolding in the presence of the native form of lysozyme. Furthermore, we believe that protein refolding is to a certain degree template-driven.

The chloroplast signal recognition particle is a heterodimeric complex of the 54kDa cytosolic signal recognition particle homologue (cpSRP54), and a novel 43kDa subunit (cpSRP43). While a nearly complete three-dimensional structure of cpSRP43 has been obtained, no structure is yet available for cpSRP54. The three-dimensional structure for cpSRP54 could provide valuable information for the rationalization of the extensive information already available regarding its function, and in the understanding of the as yet undetermined mechanism of light harvesting chlorophyll binding protein's (LHCP) insertion into the thylakoid membrane. In this study, we developed an in silico, three-dimensional structural model of cpsRP54 using a combination of homology modeling, de novo structure prediction and molecular dynamics simulation. The resulting structure is consistent with the known properties of the protein and sheds new light on some of the mechanistic details of its functioning.

Human acidic fibroblast growth factor (hFGF-1) belongs to a superfamily of 22 fibroblast growth factors (FGFs) that have a high affinity to heparin. It is a 16kD protein, which is involved in many cellular functions, such as, cell proliferation, cell differentiation, angiogenesis, tumor growth, and wound healing. In wound healing, the blood coagulation cascade is triggered to clog the wound. One of the proteases that participates in wound healing is thrombin, which converts fibrinogen to fibrin. Thrombin also cleaves wild type hFGF-1 and consequently decreases its wound healing potency. Thrombin is shown to specifically cleave FGF-1 at Arg136. In this context, in the present study we have designed several site-directed mutants at Arg136 to understand the role of this residue not only on the stability of FGF-1 but also on the FGF signaling process. The results of this study will be discussed in detail.

Fibroblast Growth Factor-1 (FGF-1) is a potent angiogenic agent that is released via the non-classical protein secretion pathway. Angiogenesis, the process of formation of new blood vessels, is vital to the formation of tumors, and is also responsible for tumor metastasis, as cancer cells travel from one part of the body to another through the newly formed vessels. Export of FGF-1 is based on the Cu2+ -dependent structure of multi-protein complexes, which involves the S100A13, a Ca2+ binding protein belonging to the family of S100 protein. The goal of this study is to characterize the structure of the FGF-1/S100A13 Data will be presented analyzing the interaction between FGF-1 and an S100A13 peptide that has been designed to mimic the binding region of FGF-1 on S100A13. The binding interaction was characterized using various biophysical techniques including ITC, DSC, proteolytic digestion, and multi-dimensional NMR spectroscopy. Characterization of the binding FGF-1/S100A13 interface is expected to shed light on the molecular mechanism(s) underlying the non-classical secretion of FGF-1.