ProtSA: a web application for calculating sequence specific protein solvent accessibilities in the unfolded ensemble

Background  

The stability of proteins is governed by the heat capacity, enthalpy and entropy changes of folding, which are strongly correlated to the change in solvent accessible surface area experienced by the polypeptide. While the surface exposed in the folded state can be easily determined, accessibilities for the unfolded state at the atomic level cannot be obtained experimentally and are typically estimated using simplistic models of the unfolded ensemble. A web application providing realistic accessibilities of the unfolded ensemble of a given protein at the atomic level will prove useful.     

Theory of protein molecule self-organization. III. A calculating method for the probabilities of the secondary structure formation in an unfolded polypeptide chain

A mathematical model is developed adequately describing an unfolded polypeptide chain without long-range interactions in which fluctuating hydrogen-bonded α-helices, β-bends, fragments of helices 3₁₀, and other local structures are formed. The obtained model is a modification of a one-dimensional Ising model for a heteropolymer and allows one to determine the probability of formation of different secondary structures in various parts of a polypeptide chain, provided the whole set of structural thermodynamic parameters exists.     

Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects

Using the heat capacity values for amino acid side-chains and the peptide unit determined in the accompanying paper, we calculated the partial heat capacities of the unfolded state for four proteins (apomyoglobin, apocytochrome c, ribonuclease A, lysozyme) in aqueous solution in the temperature range from 5 to 125 degrees C, with an assumption that the constituent amino acid residues contribute additively to the integral heat capacity of a polypeptide chain. These ideal heat capacity functions of the extended polypeptide chains were compared with the calorimetrically determined heat capacity functions of the heat and acid-denatured proteins. The average deviation of the experimental functions from the calculated ideal ones in the whole studied temperature range does not exceed the experimental error (5%). Therefore, the heat-denatured state of a protein, in solutions with acidic pH preventing aggregation, approximates well the completely unfolded state of this macromolecule. The heat capacity change caused by hydration of amino acid residues upon protein unfolding was also determined and it was shown that this is the major contributor to the observed heat capacity effect of unfolding. Its value is different for different proteins and correlates well with the surface area of non-polar groups exposed upon unfolding. The heat capacity effect due to the configurational freedom gain by the polypeptide chain was found to contribute only a small part of the overall heat capacity change on unfolding.     

Prediction of natively unfolded regions in protein chain

We have shown that the ability of a protein to be in globular or in natively unfolded state (under native conditions) may be determined (besides low overall hydrophobicity and a large net charge) by such a property as the average environment density, the average number of residues enclosed at the given distance. A statistical scale of the average number of residues enclosed at the given distance for 20 types of amino acid residues in globular state has been created on the basis of 6626 protein structures. Using this scale for separation of 80 globular and 90 natively unfolded proteins we fail only in 11% of proteins (compared with 17% of errors which are observed if to use hydrophobicity scale). The present scale may be used both for prediction of form (folded or unfolded) of the native state of protein and for prediction of natively unfolded regions in protein chains. The results of comparison of our method of predicting natively unfolded regions with the other known methods show that our method has the highest fraction of correctly predicted natively unfolded regions (that is 87% and 77% if to make averaging over residues and over proteins correspondingly).     

Electron transfer in cytochrome c. Role of the polypeptide chain

Nuclear magnetic resonance relaxation studies on partially reduced solutions of Candida krusei and horse heart cytochrome c reveal diversity in their functional behavior. Electron transfer between the two oxidation states in vitro proceeds at a rate nearly an order of magnitude slower in Canadida krusei than in the horse heart protein. This difference is interpreted in terms of an active role of the polypeptide chain in the electron transfer process since the hemes in the two proteins are identical in chemical structure and similar electronically. In both cases, electrostatic repulsion between positively charged protein molecules contributes significantly to the observed free energy of activation for the electron transfer process.     

Prediction of polypeptide fragments exposed to the solvent

A method is presented to predict those polypeptide segments within a globular protein that are more likely to be exposed to the solvent. The protein amino acidic sequence is the only information needed by this new algorithm. It uses a consensus hydrophobicity scale, derived from 28 known scales, and it is based on the comparison between the average hydrophobicity of a polypeptide fragment and the average hydrophobicity expected for a segment containing the same number of residues. The latter values are pre-computed from a non-redundant set of single chain protein structural domains. The comparison between the two average values results in a t value that readily provides the prediction with a statistical significance. A jack-knife validation analysis indicates that the protein segment predicted to be the most solvent exposed is actually solvent exposed and amongst the fragments that are most exposed. The source of a stand-alone program, written in C language, that allows the prediction of the most likely solvent exposed segment in a globular protein is available from the author.     

Solvent accessibilities in glycyl, alanyl and seryl dipeptides.

Theoretical studies on glycyl-alanyl and seryl dipeptides were performed to determine the probable backbone and side-group conformations that are preferred for solvent interaction. By following the method of Lee & Richards [(1971) J. Mol. Biol. 55, 379-400], a solute molecule is represented by a set of interlocking spheres of appropriate van der Waals radii assigned to each atom, and a solvent (water) molecule is rolled along the envelope of the van der Waals surface, and the surface accessible to the solvent molecule, and hence the solvent accessibility for a particular conformation of the solute molecule, is computed. From the calculated solvent accessibilities for various conformations, solvation maps for dipeptides were constructed. These solvation maps suggest that the backbone polar atoms could interact with solvent molecules selectively, depending on the backbone conformation. A conformation in the right-handed bridge (zetaR) region is favoured for both solvent interaction and intrachain hydrogen-bonding. Also the backbone side-chain hydrogen-bonding within the same dipeptide fragment in proteins is less favoured than hydrogen-bonding between side chain and water and between side chain and atoms of other residues. Solvent accessibilities suggest that very short distorted alphaR-helical and extended-structural parts may be stabilized via solvent interaction, and this could easily be possible at the surface of the protein molecules, in agreement with protein-crystal data.     

Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains.

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine zeta 1-thrombin:human zeta 2-thrombin and human zeta 1-thrombin:bovine zeta 2-thrombin. Human zeta 1-thrombin and zeta 2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent zeta-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between iodination and the polypeptide chain composition of thyroglobulin.

Thyroglobulin was isolated from thyroid glands of normal guinea pigs and from animals treated with thiouracil. These preparations were fractionated by isopyknic centrifugation in RbCl into proteins of varying iodine content. When the disulfide bonds of these protein fractions were reduced and analyzed by polyacrylamide gel electrophoresis in Na dodecyl-SO4, t hree species were observed with molecular weights of 295,000 (A), 210,000 (B), and 110,000 (C). Species A comprised 80% of the protein in thyroglobulin of 0.04% iodine and 13% in thyroglobulin of 0.68% iodine content. Species C showed the opposite relationship, comprising 10% of the low and 70% of the high iodine thyroglobulin. Species B was relatively independent of the iodine content and represented approximately 20% of the protein. Iodine analysis of these proteins showed species A to be lowest and species C highest. It appears that the subunit composition of thyroglobulin depends on the degree of iodination and that species A should be the only one present in the absence of iodination.     

Dynamics of unfolded polypeptide chains as model for the earliest steps in protein folding

The rate of formation of intramolecular interactions in unfolded proteins determines how fast conformational space can be explored during folding. Characterization of the dynamics of unfolded proteins is therefore essential for the understanding of the earliest steps in protein folding. We used triplet-triplet energy transfer to measure formation of intrachain contacts in different unfolded polypeptide chains. The time constants (1/k) for contact formation over short distances are almost independent of chain length, with a maximum value of about 5 ns for flexible glycine-rich chains and of 12 ns for stiffer chains. The rates of contact formation over longer distances decrease with increasing chain length, indicating different rate-limiting steps for motions over short and long chain segments. The effect of the amino acid sequence on local chain dynamics was probed by using a series of host-guest peptides. Formation of local contacts is only sixfold slower around the stiffest amino acid (proline) compared to the most flexible amino acid (glycine). Good solvents for polypeptide chains like EtOH, GdmCl and urea were found to slow intrachain diffusion and to decrease chain stiffness. These data allow us to determine the time constants for formation of the earliest intrachain contacts during protein folding.     

Hydrogen-tritium exchange of the random chain polypeptide

The Hydrogen–tritium exchange character of poly-D ,L -alanine was studied in detail as a model for the hydrogen exchange behavior of the unhindered, polymeric peptide group. The random chain nature of poly-D ,L -alanine was evident in the uniformity of exchange rate of all its hydrogens and in the similarity between this rate and that of random chain poly-D ,L -lysine and other known, unhindered secondary amide groups. An equilibrium isotope effect favoring the binding of tritium over protium to the extent of 21% was measured. Specific acid and base catalysis of the exchange and the absence of detectable general catalysis were demonstrated. Apparent energy of activation is 17 kcal/mole for deprotonation, largely due to dependence of K_w on temperature, and 15 kcal/mole for protonation, which correlates with the extreme apparent pK. The hydrogen –tritium exchange half-time rate; of poly-D ,L -alamine at any pH and temperature (T: °C) is given by the equation:      

Evidence for extrusion of unfolded extracellular enzyme polypeptide chains through membranes of Bacillus amyloliquefaciens.

The production of extracellular alpha-amylase and protease by protoplasts of Bacillus amyloliquefaciens has been achieved. The production of enzymically active protease was totally dependent on a high concentration of either Mg2+, Ca2+, or spermidine, but production of active alpha-amylase was not. This cation dependence of protease production was seen immediately upon addition of lysozyme to intact cells. The cations could prevent the inactivation of protease and alter the cytoplasmic membrane configuration of protoplasts. Production of active alpha-amylase and protease by protoplasts was totally inhibited by proteolytic enzymes such as trypsin, alpha-chymotrypsin, or the organism's purified extracellular protease. The evidence suggests that these degradative enzymes act specifically on the emerging polypeptide of the extracellular enzyme and that the polypeptide emerges in a conformation different from that of the native molecule.     

Helix-coil transitions of a polypeptide in a two-component solvent

A microscopic model is considered of a helix-coil transition of polypeptides in a two-component solvent one of whose components competes for the formation of hydrogen bonds with the peptide group. It is shown that by a redetermination of the temperature parameter the model is reduced to a polypeptide chain without the solvent. Behavior of the transition parameters is calculated in relation to the solvent concentration competing for the formation of hydrogen bonds at different parameters of polypeptide-solvent interaction.     

Analysis of the two steps in polypeptide chain initiation inhibited by pactamycin.

Earlier work has shown that the inhibition by pactamycin (PM) of polypeptide chain initiation in reticulocyte extracts is associated with (1) a defect in the joining of the 60S subunit to the smaller initiation complex to form an 80S complex ("joining reaction") (Kappen, L. S., Suzuki, H., and Goldberg, I. H. (1973), Proc. Natl. Acad. Sci. U.S.A. 70, 22) and (2) a block after the synthesis of the initial dipeptide (Kappen, L. S., and Goldberg, I. H. (1973), Biochem. Biophys. Res. Commun. 54, 1083). The relative contributions of these two effects to the action of PM and their relationship to one another were evaluated in a system employing sparsomycin that permits both initiation at a certain number of initiation sites and limited oligopeptide formation without termination and release. The degree to which PM blocks the "joining reaction" and leads to the accumulation of 48S initiation complexes that either remain free or are bound to polysomes without the corresponding 60S subunit ("half-mers") was estimated by treatment of polysomes with RNase. Met-tRNAfMet binding factors are required to stabilize the RNase-generated 48S complexes. Under conditions where the initiation factor required for the "joining reaction" functions catalytically, presumably by cycling on and off initiation complexes, PM usually inhibits 80S complex formation 50-70%. Where "joining" is not limiting (presence of at least stoichiometric amounts of joining factor or high Mg2+ concentration) PM leads to the maximal accumulation of the initial dipeptide, Met-Val, in the P-site on the ribosome, indicating a block in a subsequent step in elongation. Binding studies with [3H]PM and the inability of PM to inhibit elongation of preformed Met-Val indicate that PM must interact with the ribosomes at an early stage of initiation. Taken together these data are compatible with the suggestion that PM does not interfere with the ribosomal "joining reaction" per se, but prevents the release and reuse of the joining factor, and in so doing blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome.