Proscan: a structure-based proline design web server.

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.

Increasing Protein Conformational Stability by Optimizing β-Turn Sequence

Many proteins change their conformation upon ligand binding. For instance, bacterial periplasmic binding proteins (bPBPs), which transport nutrients into the cytoplasm, generally consist of two globular domains connected by strands, forming a hinge. During ligand binding, hinge motion changes the conformation from the open to the closed form. Both forms can be crystallized without a ligand, suggesting that the energy difference between them is small. We applied Simplicial Neighborhood Analysis of Protein Packing (SNAPP) as a method to evaluate the relative stability of open and closed forms in bPBPs. Using united residue representation of amino acids, SNAPP performs Delaunay tessellation of the protein, producing an aggregate of space-filling, irregular tetrahedra with nearest neighbor residues at the vertices. The SNAPP statistical scoring function is derived from log-likelihood scores for all possible quadruplet compositions of amino acids found in a representative subset of the Protein Data Bank, and the sum of the scores for a given protein provides the total SNAPP score. Results of scoring for bPBPs suggest that in most cases, the unliganded form is more stable than the liganded form, and this conclusion is corroborated by similar observations of other proteins undergoing conformation changes upon binding their ligands. The results of these studies suggest that the SNAPP method can be used to predict the relative stability of accessible protein conformations. Furthermore, the SNAPP method allows delineation of the role of individual residues in protein stabilization, thereby providing new testable hypotheses for rational site-directed mutagenesis in the context of protein engineering.

Apparently contradictory statements about the thermodynamics of aqueous protein solutions and of hydrophobic effect are quoted and discussed. Some credibility is found in the divergent points of view and it is pointed out that they focus attention on different aspects of the complicated conditions in aqueous solutions, some of which are more important than others for the stability of protein conformations.

The relationship between protein conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes was investigated employing alpha- and gamma-chymotrypsin. As a measure of the conformational stability the melting temperature T(m) was determined in distilled water at various pH values. The proteins were then lyophilized from those pH values where the conformational stability was maximum (pH 4.5) and minimum (pH 7.8). Protein secondary structure was quantitatively determined utilizing Fourier-transform infrared spectroscopy employing two regions sensitive to protein structure, the amide-I (1600-1700 cm(-1)) and amide-III (1215-1335 cm(-1)). Lyophilization induced significant structural alterations in both proteins, characterized by a slight decrease in the alpha-helix and a significant increase in the beta-sheet content. However, regardless of the pH from which the proteins were lyophilized, the secondary structures in the solid state were indistinguishable. This result shows that there is no relationship between the conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes. We also investigated whether lyoprotectants could minimize lyophilization-induced structural changes by increasing protein conformational stability in aqueous solution. After having identified trehalose as being efficient in largely preventing lyophilization-induced structural alterations, we conducted co-lyophilization experiments from various pH values. The results obtained exclude any contribution from increased protein conformational stability caused by the additive in aqueous solution to the beneficial structural preservation upon lyophilization. This can be understood because the dehydration and not the freezing process, as shown in an air-drying experiment, mainly causes protein structural alterations.

Arginine 54 (R54) in αA-Crystallin (αA-Cry) is highly conserved within different species. Recently, three missense mutations at this hot spot position have been reported to cause congenital cataract disorders. To investigate the impact of charge on structural and functional aspects of αA-Cry, R54 was individually substituted with lysine and aspartate. Replacement of R54 with the positively and negatively charged residues led to structural alteration and reduction in the protein conformational and proteolytic stability. Also, these mutations resulted in important increase in the amyloidogenic propensity of αA-Cry. Additionally, all these changes were more pronounced upon R54D mutation. Keeping the positive charge by R54K mutation, the structural integrity and stability of αA-Cry were partially preserved. Our results suggest that arginine 54 may also participate in salt bridge formation and conformational stabilization of αA-Cry. Also, it seems that unique physicochemical properties of arginine 54 may have a prominent role in the structural integrity, conformational stability and functional aspects of human αA-Cry.

Mechanisms of metal ion-mediated quality regulation in rehydrated Salmon: Influence on texture, protein properties, and microstructure.

Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular cross-link. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen.

Chapter 2 - Structure-Based Design for High-Hanging Vaccine Fruits

To explore the effect of an external electrostatic field (EEF) on the stability of protein conformations, the molecular dynamic modeling approach was applied to evaluate the effect of an EEF along the x or y direction on a water cluster containing a parallel or antiparallel β sheet structure. The β sheet structure contained two strands with a (Gly)3 sequence separated by a distance d along the x direction. The mean forces between the two strands along the x direction were computed from the trajectories of molecular dynamics simulations. In the absence of the EEF, the forces between the two strands in vacuum were repulsive and attractive in the parallel and antiparallel β sheet structures, respectively. In contrast, the mean forces between the two strands in water were attractive in both the parallel and antiparallel β sheet structures. This is because the electric interactions between the two strands were shielded by water, and the hydrophobic effect dominated the interaction between the two strands. When an EEF >50 MV/cm was applied to the water cluster, the attractive force between the two strands in the parallel and antiparallel β sheet structures decreased and increased, respectively. Further, the binding affinity between the two strands in the parallel and antiparallel β sheet structures also decreased and increased, respectively. This is because the large EEF leads to dielectric saturation, and consequently reduces the effects of the dielectric shielding and hydrophobic interactions.

Cas9 and Cas12a are RNA-guided endonucleases that are components of the CRISPR-Cas [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated] bacterial immune system. Cas9 and Cas12a bind to cognate CRISPR-RNAs (crRNAs) that enable them to sequence-specifically target and cleave double-stranded DNA. The binding of crRNA also triggers conformational changes in Cas9 and Cas12a that are essential for sequence-specific cleavage of the DNA at the site of crRNA- DNA pairing. Despite the accelerated use of Cas9 and Cas12a in genomic applications, including gene therapy and molecular diagnostics, they both suffer from off-target DNA cleavage where DNA that is partially complementary to the crRNA is cleaved. Removal of off-target DNA cleavage is critical for error-proof genome tools, and creation of protein variants and crRNA modifications are active areas of research in the CRISPR field. Cas9 and Cas12a are bi-lobed, multi-domain proteins. The two lobes are bridged by a highly conserved and arginine-rich helix called the bridge helix (BH). Previous studies in the field have established that BH and its conserved arginines are indispensable for target DNA cleavage. Comparison of the structures of the BHs of apo- and crRNA-DNA-bound Cas9 and Cas12a showed loop- to-helical conformational changes in both proteins. To assess the essentiality of this helical transition of the BH, we introduced proline substitutions in the BHs of Streptococcus pyogenes Cas9 (SpyCas9) and Francisella novicida Cas12a (FnoCas12a). In SpyCas9, a double proline substituent, SpyCas9-L64P/K65P (SpyCas92Pro), displayed similar cleavage of on-target DNA as the wild-type SpyCas9 (SpyCas9WT), while displayed severe reduction in cleaving off-target DNA. This feature is conserved in a double proline substituent of FnoCas12a, FnoCas12a-K969P-D970P (FnoCas12aKD2P), where BH modification provided enhanced off-target discrimination. Interestingly, FnoCas12aKD2P also displayed significant reduction in non-specific single-stranded DNA cleavage, an activity called as trans cleavage. These studies indicate that BH-conformational changes can be used as a simple and universal method for increasing the fidelity of several Cas protein families. To gain mechanistic insights of the BH-mediated off-target DNA cleavage discrimination, we are currently pursuing biochemical and structural studies. We introduced alanine substitutions instead of prolines in the BHs of SpyCas92Pro and FnoCas12aKD2P. The rationale is to test the effect of a stable helical conformation imparted by the alanine substitution, instead of the deformity that prolines can introduce in helices. Available results show that alanine substitutions reduce the off-target discrimination potential in SpyCas9 and FnoCas12a. In SpyCas9, alanine substitutions increase the rate of DNA cleavage compared to SpyCas9WT implicating that a stable helix may contribute to faster conformational transitions but reduces the ability to discriminate on-and off-target DNA substrates. On the other hand, proline substitutions seem to slowdown the conformational transitions such that an on-target DNA substrate can successfully reach the DNA cleavage competent state, but this is inhibited when bound to off-target DNA. These initial data indicate that BH can act as an allosteric sensor of DNA-RNA base pairing and change conformational transitions needed for DNA cleavage. Of note is the fact that arginine-rich helices of other RNA binding enzymes were reported to undergo conformational transitions after binding to RNA, establishing helix-ordering as an important conformational check point towards activity. To understand the structural basis of BH-mediated conformational transitions, we used cryo electron microscopy (cryo-EM) to determine the structure of FnoCas12aKD2P bound to crRNA-DNA. We faced challenges in preparing grids with good particle distribution due to the particles sticking to the carbon rather than dispersing on the holes of the cryo-grids. After testing several conditions, we identified that graphene oxide-coated grids improve particle distribution on the holes. A dataset was collected using samples dispersed on a graphene oxide-coated grid that created an initial map at 3Å resolution. Further analysis revealed conformational flexibility of certain domains of FnoCas12aKD2P-crRNA-DNA complex. We performed 3D variability analysis of the 3Å map which provided six different maps ranging in resolution from 3.2Å to 4.0 Å that represent structures of the complex at different states in the conformational transitions needed for DNA cleavage. Our results provide molecular mechanisms of BH- mediated conformational control in DNA cleavage exhibited by Cas9 and Cas12a, that can be broadly applied to other BH- containing Cas enzymes and arginine-rich helix containing RNA-binding proteins.

<sec><p indent="0mm">AlphaFold2 (AF2), the first deep learning-based protein structure prediction model to achieve near-experimental accuracy, has revolutionized the field of structural biology. With its ability to predict protein structures at unprecedented levels of accuracy, AF2 has become an essential tool in structural biology. In parallel, the deep learning methodologies derived from AF2 have made significant advancements in a range of downstream applications, such as efficient sampling of protein conformational ensembles, protein sequence design, and ligand generation. These applications are underpinned by an accurate description of the protein-free energy landscape, which is critical for modeling protein behavior in various biological contexts. </sec><sec> Despite these advancements, challenges remain, particularly in the area of multi-conformation sampling. Traditional methods such as molecular dynamics (MD) and Monte Carlo (MC) simulations, which are based on Boltzmann statistics, can provide detailed insights into conformational distributions. However, the application of these methods is hindered by their high computational cost and limited efficiency, especially for large or complex systems. In contrast, existing generative deep learning models can rapidly generate protein conformations, but often struggle to incorporate biophysical constraints, and are limited in the power of predicting the accurate energy minima, avoiding steric clashes, or understanding physics such as electrostatic interactions. This lack of control over the physical space makes it difficult to generate conformations that are both physically realistic and biologically relevant. </sec><sec> Furthermore, the robustness of the initial protein structure is crucial for downstream applications, including protein-protein interaction predictions, molecular docking, and virtual screening, as well as reliable and efficient MD simulations. However, conventional structure repair tools often fail to accurately model missing regions, such as loops or partially resolved regions, which may lead to unrealistic geometries or energetically unfavorable conformations. </sec><sec> To address these limitations, we present FoldCopilot, an innovative suite based on AF2. FoldCopilot uses both target sequences and multiple sequence alignments (MSAs) as input prompts for the Evoformer module, leveraging a homology-based sequence perturbation mechanism that generates diverse local conformations without the need for retraining. This method allows for the efficient generation of protein conformations and at the same time maintains high fidelity with known structural templates and evolutionary constraints. In addition, FoldCopilot integrates structural templates and initial reference frames into the iterative refinement process, enabling the directed evolution and control over generated protein structures. This iterative process ensures that the generated conformations not only adhere to biophysical principles, such as realistic energy states and steric compatibility but also meet specific functional requirements dictated by the target application. </sec><sec> Our experimental results demonstrate that FoldCopilot excels in key tasks such as protein structure repair and localized multi-conformation generation. The method significantly outperforms traditional structure repair techniques, yielding more accurate and biologically relevant models for regions of missing data. Additionally, FoldCopilot shows a high capability to generate diverse conformations, making it an ideal tool for protein engineering, protein-ligand interaction predictions, and other structure-based drug design applications. </sec><sec> In summary, FoldCopilot bridges the gap between high-fidelity conformational sampling and the ability to control conformational space, offering a powerful approach to facilitate protein-molecule interaction predictions, structural modeling, and molecular dynamics simulations. </sec>

Protein folding is an exploding area of research in biophysics and physical chemistry. Here, we describe the integration of several techniques, including absorption spectroscopy, fluorescence spectroscopy, and Förster resonance energy transfer (FRET) measurements, to probe important topics in protein folding. Cytochrome c is used as a model protein; comparison of conformational stabilities ( ΔGH2O∘) measured via two chemical denaturants, urea and guanidinium hydrochloride, illustrate important concepts in protein folding and intermolecular interactions. In addition, the determination of intraprotein distances based upon the FRET pair Trp-59 and the heme group for unfolded states of cytochrome c highlights the evolution of the protein structure under unfolding conditions. Analysis and discussion of these results provide opportunities to gain in-depth understanding of models for protein folding while enhancing students' skills with optical techniques. Collectively, the combination of optical spectroscopy, rigorous quantitative analysis, and a focus on biophysics illustrates the significance of fundamental research at the growing intersection of chemistry, biology, and physics.

Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1–1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1–542) or intermediate (TcdA1102–1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.

With the rapid growth of structural data in the Protein Data Bank, efficient mining and utilization of protein-ligand interaction pattern information from these structures can advance rational drug design. Here, we present SiteMatcher, a web server that integrates a curated repository of protein-ligand interaction motifs from the Protein Data Bank with an automated fragment-grafting engine to facilitate structure-based ligand design. The engine identifies motifs similar to user-defined local protein sites, extracts ligand fragments from these motifs, and merges them─either directly or via suitable linkers─with the seed compound to produce ligands complementary to the target pocket. By leveraging site similarity-based fragment reuse, SiteMatcher helps users explore diverse ligand design strategies efficiently. We hope that SiteMatcher will serve as a practical tool for medicinal chemists. The server is freely available at https://sitematcher.xundrug.cn.