Evidence for a strong selenium-aromatic interaction derived from crystallographic data and ab initio quantum chemical calculations

Increasing attention is being paid to the role of selenium, both as an essential component required for the activity of many enzymes and in the context of selenium-based pharmaceutical agents. A wide range of therapeutics that include selenium are on the market and under development, such as antihypertensive, anticancerogenic, antiviral, and immunosuppressive agents. Computer-aided drug design (CADD) has proven to be an important tool for the development of new drugs. Many CADD techniques, including docking, molecular dynamics simulation, and other receptor-based approaches, require an accurate understanding of the nature of the intermolecular forces that act to stabilize protein-ligand complexes; moreover, a quantitative assessment of these interactions furthers our efforts to rationalize the drug design process. In this paper, we consider one class of interaction involving selenium, that between Se and aromatic rings. Prior work has shown that interactions between divalent sulfur and aromatic rings are observed much more frequently than would be expected on the basis of chance, both in protein structures and the crystal structures of organic compounds that include these moieties. Recent studies on the optimization of inhibitor-protein binding also suggest that sulfur-aromatic interactions are important in stabilizing these complexes and may be crucial focal point for CADD. Given that selenium and sulfur have similar chemistry, and that selenium is significantly more polarizable, we propose that Se-aromatic interactions may also play an important stabilizing role in the structure of folded proteins and in drug-protein complexes. We have tested this hypothesis against data from the Cambridge Crystallographic Database and ab initio quantum chemical calculations. We have found evidence that selenium does interact strongly with aromatic rings and may play a role analogous to sulfur in stabilizing protein folds. In addition, selenium should be considered along with sulfur in rational drug design strategies that seek to improve binding to target protein sites that include aromatic rings.     

First-step mutations for adaptation at elevated temperature increase capsid stability in a virus

The relationship between mutation, protein stability and protein function plays a central role in molecular evolution. Mutations tend to be destabilizing, including those that would confer novel functions such as host-switching or antibiotic resistance. Elevated temperature may play an important role in preadapting a protein for such novel functions by selecting for stabilizing mutations. In this study, we test the stability change conferred by single mutations that arise in a G4-like bacteriophage adapting to elevated temperature. The vast majority of these mutations map to interfaces between viral coat proteins, suggesting they affect protein-protein interactions. We assess their effects by estimating thermodynamic stability using molecular dynamic simulations and measuring kinetic stability using experimental decay assays. The results indicate that most, though not all, of the observed mutations are stabilizing.     

Recognition and interactions controlling the assemblies of beta barrel domains.

We present a qualitative computer graphics approach to the characterization of forces important to the assembly of beta domains that should have general utility for examining protein interactions and assembly. In our approach, the nature of the molecular surface buried by the domain contacts, the specificity of the residue-to-residue interactions, and the identity of electrostatic, hydrophobic, and hydrophilic interactions are elucidated. These techniques are applied to the beta barrel domains of Cu, Zn superoxide dismutase (SOD), immunoglobulin Fab, and tomato bushy stunt virus coat protein (TBSV), a plant viral capsid protein. By looking at a set of proteins having different numbers of interacting beta domains, we have been able to see some of the variety and also some of the patterns common to these assembled domains. Strong beta domain interactions (identified by their biochemical integrity) are apparently due to chemical, electrostatic, and shape complementarity of the molecular surfaces buried from interaction with solvent molecules. Although the amount of hydrophobic buried surface area appears to correlate with the strength of the interaction, electrostatic forces appear to be important in both stabilizing and destabilizing specific contacts. In TBSV, analysis of electrostatic interactions may help explain mechanisms of subunit accommodation to different environments, particle expansion, and pathways of assembly. The possible molecular basis for observed differences in the stability and flexibility of the domain complexes is discussed.     

Molecular interactions between a plant virus movement protein and RNA: force spectroscopy investigation

RNA-protein interactions are fundamental for different aspects of molecular biology such as gene expression, assembly of biomolecular complexes or macromolecular transport. The 3a movement protein (MP) of a plant virus, Cucumber mosaic virus (CMV), forms ribonucleoprotein (RNP) complexes with viral RNA, capable of trafficking from cell-to-cell throughout the infected plant only in the presence of the CMV capsid protein (CP). However, deletion of the C-terminal 33 amino acid residues of the CMV MP (in the mutant designated 3aDeltaC33 MP) resulted in CP-independent cell-to-cell movement. The biological differences in the behaviour of CMV wild type (wt) 3a MP and 3aDeltaC33 MP could have been a consequence of differences in the RNA-binding properties of the two MPs detected previously using biochemical assays on ensembles of molecules. To investigate the physical mechanisms of MP-RNA interactions at a single molecule level, we applied atomic force microscopy to measure for the first time unbinding forces between these individual binding partners. Minimal unbinding forces determined for individual interaction of the CMV RNA molecule with the CMV wt or truncated MPs were estimated to be approximately 45 pN and approximately 90 pN, respectively, suggesting that the distinct differences in the strength of MP-RNA interactions for the wt MP and truncated MP are attributable to the molecular binding mechanism. We also demonstrated that molecules of both CMV 3a MP and 3aDeltaC33 MP were capable of self-interaction with minimal unbinding forces of approximately 50 pN and approximately 70 pN, respectively, providing a physical basis for the cooperative mechanism of the RNA binding. The significance of intermolecular force measurements for understanding the structural and functional aspects of viral RNP formation and trafficking is discussed.     

On the temperature dependence of complex formation between chitosan and proteins

Chitosan is a biocompatible easily degradable polysaccharide, which, because of its positive charge, is able to interact favorably with deprotonated carboxyl groups of proteins. The strength of these charge-charge interactions is generally low, resulting in poor colloidal stability of the complexes. To investigate if other noncovalent forces contribute to stabilizing such systems, we have selected α-lactalbumin, β-lactoglobulin, β-casein, and human growth hormone, characterized by a common acidic pI value (～ 5) that ensures their overall negative charge at physiological pH. Binding energetics between chitosan and proteins was studied by isothermal titration calorimetry, whereas the thermal stability was assessed by differential scanning calorimetry. Our data show that colloidal stability of the particles depends on protein identity as well as temperature, indicating the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) as driving forces for the complex formation. This suggests that chitosan-protein drug delivery systems can be improved through preparation process optimization with regard to temperature.     

Interpreting protein/DNA interactions: distinguishing specific from non-specific and electrostatic from non-electrostatic components

We discuss the effectiveness of existing methods for understanding the forces driving the formation of specific protein-DNA complexes. Theoretical approaches using the Poisson-Boltzmann (PB) equation to analyse interactions between these highly charged macromolecules to form known structures are contrasted with an empirical approach that analyses the effects of salt on the stability of these complexes and assumes that release of counter-ions associated with the free DNA plays the dominant role in their formation. According to this counter-ion condensation (CC) concept, the salt-dependent part of the Gibbs energy of binding, which is defined as the electrostatic component, is fully entropic and its dependence on the salt concentration represents the number of ionic contacts present in the complex. It is shown that although this electrostatic component provides the majority of the Gibbs energy of complex formation and does not depend on the DNA sequence, the salt-independent part of the Gibbs energy--usually regarded as non-electrostatic--is sequence specific. The CC approach thus has considerable practical value for studying protein/DNA complexes, while practical applications of PB analysis have yet to demonstrate their merit.     

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins: ROLES IN PROTEIN FOLDING,STABILITY, AND FUNCTION*

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.     

Stacking and hydrogen bond interactions between adenine and gallic acid

We have performed DFT and DFT-SAPT calculations on dimers of gallic acid, the model system for plant polyphenols, and the DNA base adenine. These dimers were selected for this study as they exhibit simultaneously hydrogen bonds and stacking interactions and it allows to quantify the relative values of these interactions. We calculate the relationships between the stability of the complexes, the charge transfer between monomers and the properties of the intermolecular bonds including hydrogen bonds and other bonds that do not involve hydrogen atoms. DFT-SAPT calculations were also performed to obtain the participation of the different types of energy and so the resulting physical effects. The results show that the presence of hydrogen bonds is the main stabilizing factor for dimers: the higher number and strength, the lower the dimer energy. The contribution of stacking to the stabilization is related to the strength and number of bonds between non-hydrogen atoms and quantified by the contribution of the dispersion terms to the interaction energy. Dimers I and II are mainly stabilized due to hydrogen bonds whereas dimer III is mainly stabilized by stacking interactions.     

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.     

Amyloid-forming propensity of the hydrophobic non-natural amino acid on the fibril-forming core peptide of human tau

Amino acid residues with aromatic side chains, such as Tyr and Phe, are known to play essential roles in forming and stabilizing the amyloid fibrils of pathogenic polypeptides by affecting their amyloid forming propensity. We have studied the amyloid-type aggregation of peptides containing non-natural amino acid derived from a core part of human pathogenic protein, tau. The hydrophobic nature of the biphenyl group and its intermolecular aromatic interactions strongly alter their amyloid formation properties.     

Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy

Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.     

Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton's tyrosine kinase

The SH3 domain of Bruton's tyrosine kinase (Btk) is preceded by the Tec homology (TH) region containing proline-rich sequences. We have studied a protein fragment containing both the Btk SH3 domain and the proline-rich sequences of the TH region (PRR-SH3). Intermolecular NMR cross-relaxation measurements, gel permeation chromatography profiles, titrations with proline-rich peptides, and (15)N NMR relaxation measurements are all consistent with a monomer-dimer equilibrium with a dissociation constant on the order of 60 microM. The intermolecular interactions do, at least in part, involve proline-rich sequences in the TH region. This behavior of Btk PRR-SH3 may have implications for the functional action of Btk.     

Molecular determinants for ATP-binding in proteins: a data mining and quantum chemical analysis

Adenosine 5'-triphosphate (ATP) plays an essential role in all forms of life. Molecular recognition of ATP in proteins is a subject of great importance for understanding enzymatic mechanism and for drug design. We have carried out a large-scale data mining of the Protein Data Bank (PDB) to analyze molecular determinants for recognition of the adenine moiety of ATP by proteins. Non-bonded intermolecular interactions (hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) between adenine base and surrounding residues in its binding pockets are systematically analyzed for 68 non-redundant, high-resolution crystal structures of adenylate-binding proteins. In addition to confirming the importance of the widely known hydrogen bonding, we found out that cation-pi interactions between adenine base and positively charged residues (Lys and Arg) and pi-pi stacking interactions between adenine base and surrounding aromatic residues (Phe, Tyr, Trp) are also crucial for adenine binding in proteins. On average, there exist 2.7 hydrogen bonding interactions, 1.0 pi-pi stacking interactions, and 0.8 cation-pi interactions in each adenylate-binding protein complex. Furthermore, a high-level quantum chemical analysis was performed to analyze contributions of each of the three forms of intermolecular interactions (i.e. hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) to the overall binding force of the adenine moiety of ATP in proteins. Intermolecular interaction energies for representative configurations of intermolecular complexes were analyzed using the supermolecular approach at the MP2/6-311 + G* level, which resulted in substantial interaction strengths for all the three forms of intermolecular interactions. This work represents a timely undertaking at a historical moment when a large number of X-ray crystallographic structures of proteins with bound ATP ligands have become available, and when high-level quantum chemical analysis of intermolecular interactions of large biomolecular systems becomes computationally feasible. The establishment of the molecular basis for recognition of the adenine moiety of ATP in proteins will directly impact molecular design of ATP-binding site targeted enzyme inhibitors such as kinase inhibitors.     

Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements

In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular β-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule.     

Energetics of membrane protein folding and stability

The critical role of membrane proteins in a myriad of biological and physiological functions has spawned numerous investigations over the past several decades with the long-term goal of identifying the molecular origins and energetic forces that stabilize these proteins within the membrane. Parallel structural and thermodynamics studies on several systems have provided significant insight regarding the driving forces governing folding, assembly, insertion, and translocation of membrane proteins. The present review surveys families of membrane-associated proteins including α-helical and β-barrel structures, viral surface receptors, and pore-forming toxins, citing representative proteins within each of these classes for further scrutiny in terms of structure-function relationships and global conformational stability. This overview presents seminal findings from pioneering studies on the energetics of membrane protein folding and stability to modern techniques that are exploiting the use of molecular genetics and single molecule studies. An overall consensus regarding the molecular origins of membrane protein stability is that a number of intrinsic properties resemble features of soluble proteins, yet there are distinct energetic differences arising from specific intra- and intermolecular interactions within the membrane. The combined efforts from structural, energetics, and dynamics approaches offer unique insights and improve our fundamental understanding of the driving forces dictating membrane protein folding and stability.     

Protein-DNA interactions: structural, thermodynamic and clustering patterns of conserved residues in DNA-binding proteins

Amino acid residues, which play important roles in protein function, are often conserved. Here, we analyze thermodynamic and structural data of protein-DNA interactions to explore a relationship between free energy, sequence conservation and structural cooperativity. We observe that the most stabilizing residues or putative hotspots are those which occur as clusters of conserved residues. The higher packing density of the clusters and available experimental thermodynamic data of mutations suggest cooperativity between conserved residues in the clusters. Conserved singlets contribute to the stability of protein-DNA complexes to a lesser extent. We also analyze structural features of conserved residues and their clusters and examine their role in identifying DNA-binding sites. We show that about half of the observed conserved residue clusters are in the interface with the DNA, which could be identified from their amino acid composition; whereas the remaining clusters are at the protein-protein or protein-ligand interface, or embedded in the structural scaffolds. In protein-protein interfaces, conserved residues are highly correlated with experimental residue hotspots, contributing dominantly and often cooperatively to the stability of protein-protein complexes. Overall, the conservation patterns of the stabilizing residues in DNA-binding proteins also highlight the significance of clustering as compared to single residue conservation.     

The influence of secondary interactions on complex stability and double proton transfer reaction in 2-[1H]-pyridone/2-hydroxypyridine dimers

The 2-[1H]-pyridone/2-hydroxypyridine tautomeric pair and its 6-substituted complexes have been studied with the use of DFT(M05) method. The intermolecular interaction energy has been calculated and discussed in the light of secondary interaction concept. The attractive secondary interactions of O/NH and O/OH type and OH/NH and OH/OH repulsions have been analyzed in terms of stabilizing or destabilizing influence on intermolecular behavior. The transition states of the double proton transfer reaction have been found and the energy of activation has been determined. The activation energy of the proton transfer reaction, geometry of the complexes and transition states show NH(2) and/or OH groups influence the properties of complexes and transition states. The HOMA index of aromaticity was applied to describe the π-electron delocalization in the heterocyclic rings.     

New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus

The crystal structure of the seven-iron ferredoxin from Thermus thermophilus (FdTt) has been determined at 1.64 A resolution, allowing us to unveil the common mechanisms of thermostabilization within "bacterial-type" ferredoxins. FdTt and other homologous thermophilic seven-iron ferredoxins are smaller than their mesophilic counterparts. Thermostabilizing features are optimized in a minimal structural and functional unit, with an extensive cross-linking of secondary structure elements mediated by improved polar and hydrophobic interactions. Most of the potentially stabilizing features are focused on the vicinity of the functional [3Fe-4S] cluster. The structural [4Fe-4S] cluster is shielded in thermophilic FdTt by an increased number of polar interactions involving the two N-terminal residues. Comparisons with the hyperthermostable ferredoxin from Thermotoga maritima reveal that (1) a reduction in the number of non-glycine residues in strained conformations, (2) improved polar interactions within the common iron-sulfur cluster binding (betaalphabeta)2 motif, and (3) an optimized charge distribution at the protein surface, constitute a common strategy for increasing the thermal stability of these ferredoxins.