Structural characterization of a methionine-rich, emulsifying protein from sunflower seed

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.     

"De novo" design of peptides with specific lipid-binding properties

In this study, we describe an in silico method to design peptides that can be made of non-natural amino acids and elicit specific membrane-interacting properties. The originality of the method holds in the capacities developed to design peptides from any non-natural amino acids as easily as from natural ones, and to test the structure stability by an angular dynamics rather than the currently-used molecular dynamics. The goal of this study was to design a non-natural tilted peptide. Tilted peptides are short protein fragments able to destabilize lipid membranes and characterized by an asymmetric distribution of hydrophobic residues along their helix structure axis. The method is based on the random generation of peptides and their selection on three main criteria: mean hydrophobicity and the presence of at least one polar residue; tilted insertion at the level of the acyl chains of lipids of a membrane; and conformational stability in that hydrophobic phase. From 10,000,000 randomly-generated peptides, four met all the criteria. One was synthesized and tested for its lipid-destabilizing properties. Biophysical assays showed that the "de novo" peptide made of non-natural amino acids is helical either in solution or into lipids as tested by Fourier transform infrared spectroscopy and is able to induce liposome fusion. These results are in agreement with the calculations and validate the theoretical approach.     

Comparative study on specificities of rat cathepsin L and papain: amino acid differences at substrate-binding sites are involved in their specificities

Sixty-nine rat cathepsin L-susceptible peptide bonds were analyzed employing various peptide substrates. The proteolytic specificities of rat cathepsin L and papain were compared and the results are discussed in relation to differences in amino acid residues around their binding sites. The specificity of cathepsin L, which is characterized by a remarkable preference for hydrophobic amino acids at the P2 site of the scissile peptide bonds, was analogous to that of papain as a whole. This analogous specificity suggests that the binding sites of the two proteases are analogous, as expected from their homologous amino acid sequences. However, there is a slight difference in the preference for S3 site between them. That is, cathepsin L showed a greater preference for bulky and hydrophobic amino acids at the S3 site than did papain. Based on the computer-graphically deduced structure of the binding sites of cathepsin L, the preferences for hydrophobic amino acids at the S2 site and for bulky and hydrophobic amino acids at the S3 site of the protease are supposed to be related to the compensating amino acid substitutions at the S2 site (V133A and V157L) and the reduction in size at the S3 site (Y61Q and Y67L), respectively. The discussion of the effect of the amino acid substitutions on the proteolytic activities of cathepsin L and papain in this paper provides a basis for more advanced studies of the relationship between structure and function of proteases belonging to the papain superfamily by means of protein engineering.     

Identification of a novel and potent inhibitor of phospholipase A(2) in a medicinal plant: crystal structure at 1.93Å and Surface Plasmon Resonance analysis of phospholipase A(2) complexed with berberine

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93? and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).     

A "solvated rotamer" approach to modeling water-mediated hydrogen bonds at protein-protein interfaces

Water-mediated hydrogen bonds play critical roles at protein-protein and protein-nucleic acid interfaces, and the interactions formed by discrete water molecules cannot be captured using continuum solvent models. We describe a simple model for the energetics of water-mediated hydrogen bonds, and show that, together with knowledge of the positions of buried water molecules observed in X-ray crystal structures, the model improves the prediction of free-energy changes upon mutation at protein-protein interfaces, and the recovery of native amino acid sequences in protein interface design calculations. We then describe a "solvated rotamer" approach to efficiently predict the positions of water molecules, at protein-protein interfaces and in monomeric proteins, that is compatible with widely used rotamer-based side-chain packing and protein design algorithms. Finally, we examine the extent to which the predicted water molecules can be used to improve prediction of amino acid identities and protein-protein interface stability, and discuss avenues for overcoming current limitations of the approach.     

Hydropathic anti-complementarity of amino acids based on the genetic code

An interesting pattern in the genetic code has been discovered. Codons for hydrophilic and hydrophobic amino acids on one strand of DNA are complemented by codons for hydrophobic and hydrophilic amino acids on the other DNA strand, respectively. The average tendency of codons for "uncharged" (slightly hydrophilic) amino acids is to be complemented by codons for "uncharged" amino acids.     

Mechanisms of amino acid partitioning in cationic reversed micelles

The aim of this work is to discuss the mechanisms involved in amino acidsolubilization in cationic reversed micelles. A simple mechanism was assumedin which the amino acid solubilization is mediated by an ion-exchangeprocess between the amino acid and the surfactant counter ion neglecting theeffect of the reversed micellar structure. Based on this mechanism a simplemodel to predict equilibrium was developed and applied to the solubilizationof amino acids with different structures. It was found that solubilizationof hydrophilic and slightly hydrophobic amino acids can be described by anion-exchange mechanism and the amino acid equilibrium concentration can bedetermined for different experimental conditions using this model. However,solubilization of hydrophobic amino acids can not be described by a simpleion-exchange model. In this case hydrophobic contributions play an importantrole in amino acid solubilization and must be considered in the overallsolubilization process. This hydrophobic contribution was evaluated bydetermination of an interfacial partition coefficient. The overall aminoacid extraction was determined using distribution coefficients of all theamino acid forms and considering their dependence on ionic strength.     

Membrane bound pituitary metalloendopeptidase: Apparent identity to enkephalinase

A membrane bound zinc-metalloendopeptidase from bovine pituitaries with a specificity toward bonds on the amino side of hydrophobic amino acids, cleaves Met- and Leu-enkephalin at the Gly-Phe bond, releasing Phe-Met and Phe-Leu respectively. The enzyme also hydrolyzes bonds on the amino side of hydrophobic amino acids in oxytocin, bradykinin, neurotensin and several synthetic substrates. A free carboxyl group on a dipeptide C-terminal to the hydrolyzed bond is not a requirement for activity. The enzyme is also present in brain membrane fractions. The regional distribution of this enzyme in brain, its specificity toward natural and synthetic substrates, and its sensitivity to inhibitors, suggest that the enzyme is identical to an activity referred to as “enkephalinase”, which has been described as dipeptidyl carboxypeptidase. The data show that the enzyme is an endopeptidase with a specificity similar to that of a group of microbial proteases, one of which is thermolysin.     

Selective activation of the 20 S proteasome (multicatalytic proteinase complex) by histone h3.

Two distinct activities cleaving bonds after hydrophobic amino acids have been identified in the bovine pituitary 20 S proteasome. One, expressed by the X subunit, that cleaves bonds after aromatic and branched chain amino acids was designated as chymotrypsin-like (ChT-L).(1) The second, expressed by the Y subunit, that cleaves bonds after acidic amino acids was designated as peptidylglutamyl-peptide hydrolyzing (PGPH) but also cleaves bonds after branched chain amino acids. Low micromolar concentrations of the arginine-rich histone H3 (H3) are shown to induce changes in the specificity of the proteasome by selectively activating cleavages after branched chain and acidic amino acids while inhibiting cleavage of peptidyl-arylamide bonds in synthetic substrates. H3 activates 15-fold cleavage after leucine but not phenylalanine residues in model synthetic substrates. The activation is associated with a decrease in K(m) and an increase in V(max), suggesting positive allosteric activation. H3 activates more than 60-fold degradation of the oxidized B-chain of insulin, by cleaving mainly bonds after acidic and branched chain amino acids, and accelerates the degradation of casein and lysozyme, the latter in the presence of dithiothreitol. The degradation of lysozyme in the presence of H3 generates fragments that differ from those in its absence, indicating H3-induced specificity changes. H3 inhibits cleavage of the Trp3-Ser4 and Tyr5-Gly6 bonds in gonadotropin releasing hormone, bonds cleaved by the ChT-L activity in the absence of H3. The results suggest H3-selective activation of the Y subunit and specificity changes that could potentially affect proteasomal function in the nuclear compartment.     

Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations

Understanding the solvation of amino acids in biomembranes is an important step to better explain membrane protein folding. Several experimental studies have shown that polar residues are both common and important in transmembrane segments, which means they have to be solvated in the hydrophobic membrane, at least until helices have aggregated to form integral proteins. In this work, we have used computer simulations to unravel these interactions on the atomic level, and classify intramembrane solvation properties of amino acids. Simulations have been performed for systematic mutations in poly-Leu helices, including not only each amino acid type, but also every z-position in a model helix. Interestingly, many polar or charged residues do not desolvate completely, but rather retain hydration by snorkeling or pulling in water/headgroups--even to the extent where many of them exist in a microscopic polar environment, with hydration levels corresponding well to experimental hydrophobicity scales. This suggests that even for polar/charged residues a large part of solvation cost is due to entropy, not enthalpy loss. Both hydration level and hydrogen bonding exhibit clear position-dependence. Basic side chains cause much less membrane distortion than acidic, since they are able to form hydrogen bonds with carbonyl groups instead of water or headgroups. This preference is supported by sequence statistics, where basic residues have increased relative occurrence at carbonyl z-coordinates. Snorkeling effects and N-/C-terminal orientation bias are directly observed, which significantly reduces the effective thickness of the hydrophobic core. Aromatic side chains intercalate efficiently with lipid chains (improving Trp/Tyr anchoring to the interface) and Ser/Thr residues are stabilized by hydroxyl groups sharing hydrogen bonds to backbone oxygens.     

Hydrophobic residues can identify native protein structures

Evaluation of protein structures needs a trustworthy potential function. Although several knowledge‐based potential functions exist, the impact of different types of amino acids in the scoring functions has not been studied yet. Previously, we have reported the importance of nonlocal interactions in scoring function (based on Delaunay tessellation) in discrimination of native structures. Then, we have questioned the structural impact of hydrophobic amino acids in protein fold recognition. Therefore, a Hydrophobic Reduced Model (HRM) was designed to reduce protein structure of FS (Full Structure) into RS (Reduced Structure). RS is considered as a reduced structure of only seven hydrophobic amino acids (L, V, F, I, A, W, Y) and all their interactions. The presented model was evaluated via four different performance metrics including the number of correctly identified natives, the Z‐score of the native energy, the RMSD of the minimum score, and the Pearson correlation coefficient between the energy and the model quality. Results indicated that only nonlocal interactions between hydrophobic amino acids could be sufficient and accurate enough for protein fold recognition. Interestingly, the results of HRM is significantly close to the model that considers all amino acids (20‐amino acid model) to discriminate the native structure of the proteins on eleven decoy sets. This indicates that the power of knowledge‐based potential functions in protein fold recognition is mostly due to hydrophobic interactions. Hence, we suggest combining a different well‐designed scoring function for non‐hydrophobic interactions with HRM to achieve better performance in fold recognition.     

Hydrophobic pockets at the membrane interface: an original mechanism for membrane protein interactions

The effect of partial digestion by trypsin and GluC protease on the association of the membrane polypeptides of LH1 from Rhodospirillum (Rsp.) rubrum was studied. Trypsin and GluC protease treatments of LH1 result in the cleavage of the first three amino acids from the alpha polypeptide and of the first 18 amino acids from the beta polypeptide, respectively, without any noticeable reorganization of their secondary structure, as measured by attenuated total reflectance Fourier transform IR spectroscopy. However, the enthalpy variation accompanying dimer formation was dramatically reduced by the protease attacks by as much as 80%. Our results show that the alphabeta heterodimer is mainly stabilized by hydrophobic interactions which involve the amino-terminal extensions of the participating polypeptides. Using the close homology between the polypeptides of Rsp. rubrum LH1 and that of Rsp. molischianum LH2, whose structure is known, a structural model for these "hydrophobic pockets" lying close to the membrane interface is proposed.     

Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region

The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin-binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 A resolution in the space group P2(1) with two protein-ligand complexes in the asymmetric unit. In both molecules, the characteristic beta-barrel architecture with the attached alpha-helix is well preserved. In contrast, the four loops at one end of the beta-barrel that form the entrance to the binding site exhibit large conformational deviations from the wild-type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IX(gamma) in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the beta-barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so-called "anticalins" directed against prescribed targets as an alternative to recombinant antibody fragments.     

Design and characterization of a membrane permeable N-methyl amino acid-containing peptide that inhibits Abeta1-40 fibrillogenesis.

Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of beta-sheet containing fibrils. In a previous publication, we demonstrated that the self-association of the Alzheimer's beta-amyloid (Abeta) peptide is inhibited by peptides homologous to the central core domain of Abeta, but containing N-methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, beta-strand conformation, the alternating position of N-methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen-bonding capabilities due to the replacement of the amide protons by N-methyl groups. Here, we demonstrate, through two-dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N-methyl amino acids, Abeta16-20m or Ac-K(Me)LV(Me)FF-NH2, does indeed have the intended structure of an extended beta-strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine-HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm, in contrast to the nonmethylated congener, Abeta16-20 (Ac-KLVFF-NH2). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Abeta16-20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through beta-strand domains.     

Chemical modification of proteins by "smart" polymers

The modification of proteinase inhibitor ovomucoid from duck eggs white by poly-N,N-diethylacrylamide having a low critical solution temperature (LCST) have been studied. Modification of free amino groups of lysine and N-terminal residue of ovomucoid is resulted in a significant decrease in the activity of the inhibitor toward trypsin and small decrease in the activity toward α-chymotrypsin. At heating of the solution of modified ovomucoid above the LCST transformation of the antitryptic centers of ovomucoid in antichymotryptic centers was observed. It was shown that this phenomenon is due to the hydrophobization the lysine residues localized in the reactive centers of the inhibitor while maintaining the structure of the "linkage loops". Therefore the α-chymotrypsine molecules began to interact with these residues, mistaking them for the residues of hydrophobic amino acids of antichymotryptic centers.     

Unusual specificity of polyethylene glycol-modified thermolysin in peptide synthesis catalyzed in organic solvents

Summary Polyethylene glycol-modified thermolysin was found to efficiently catalyze peptide synthesis in organic solvents. As in aqueous media, the reaction occurred through a rapid equilibrium random bireactant mechanism. However, the substrate specificity of modified thermolysin was actually changed since hydrophilic as well as acidic amino acids were better carboxyl group donors than hydrophobic residues, contrary to what is observed in both the enzyme-catalyzed synthesis and hydrolysis of peptide bonds in water.     

Sequence periodicity and secondary structure propensity in model proteins

We explore the question of whether local effects (originating from the amino acids intrinsic secondary structure propensities) or nonlocal effects (reflecting the sequence of amino acids as a whole) play a larger role in determining the fold of globular proteins. Earlier circular dichroism studies have shown that the pattern of polar, non polar amino acids (nonlocal effect) dominates over the amino acid intrinsic propensity (local effect) in determining the secondary structure of oligomeric peptides. In this article, we present a coarse grained computational model that allows us to quantitatively estimate the role of local and nonlocal factors in determining both the secondary and tertiary structure of small, globular proteins. The amino acid intrinsic secondary structure propensity is modeled by a dihedral potential term. This dihedral potential is parametrized to match with experimental measurements of secondary structure propensity. Similarly, the magnitude of the attraction between hydrophobic residues is parametrized to match the experimental transfer free energies of hydrophobic amino acids. Under these parametrization conditions, we systematically explore the degree of frustration a given polar, non polar pattern can tolerate when the secondary structure intrinsic propensities are in opposition to it. When the parameters are in the biophysically relevant range, we observe that the fold of small, globular proteins is determined by the pattern of polar, non polar amino acids regardless of their instrinsic secondary structure propensities. Our simulations shed new light on previous observations that tertiary interactions are more influential in determining protein structure than secondary structure propensity. The fact that this can be inferred using a simple polymer model that lacks most of the biochemical details points to the fundamental importance of binary patterning in governing folding.