Analysis and prediction of protein folding rates using quadratic response surface models

Understanding the relationship between amino acid sequences and folding rates of proteins is an important task in computational and molecular biology. In this work, we have systematically analyzed the composition of amino acid residues for proteins with different ranges of folding rates. We observed that the polar residues, Asn, Gln, Ser, and Lys, are dominant in fast folding proteins whereas the hydrophobic residues, Ala, Cys, Gly, and Leu, prefer to be in slow folding proteins. Further, we have developed a method based on quadratic response surface models for predicting the folding rates of 77 two- and three-state proteins. Our method showed a correlation of 0.90 between experimental and predicted protein folding rates using leave-one-out cross-validation method. The classification of proteins based on structural class improved the correlation to 0.98 and it is 0.99, 0.98, and 0.96, respectively, for all-alpha, all-beta, and mixed class proteins. In addition, we have utilized Baysean classification theory for discriminating two- and three-state proteins, which showed an accuracy of 90%. We have developed a web server for predicting protein folding rates and it is available at http://bioinformatics.myweb.hinet.net/foldrate.htm.     

Delivering Structural Information on the Polar Face of Membrane‐Active Peptides: 19F‐NMR Labels with a Cationic Side Chain

Conformationally constrained non‐racemizing trifluoromethyl‐substituted lysine isosteres [(E)‐ and (Z)‐TCBLys] with charged side chains are presented as a new type of ¹⁹F‐NMR labels for peptide studies. Design of the labels, their synthesis, incorporation into peptides and experimental demonstration of their application for solid state NMR studies of membrane‐active peptides are described. A series of fluorine‐labeled analogues of the helical amphipathic antimicrobial peptide PGLa(Nle) was obtained, in which different lysine residues in the original peptide sequence were replaced, one at a time, by either (E)‐ or (Z)‐TCBLys. Antimicrobial activities of the synthesized analogues were practically the same as those of the parent peptide. The structural and orientational parameters of the helical PGLa(Nle) peptide in model bilayers, as determined using the novel labels confirmed and refined the previously known structure. (E)‐ and (Z)‐TCBLys, as a set of cationic ¹⁹F‐NMR labels, were shown to deliver structural information about the charged face of amphipathic peptides by solid state ¹⁹F‐NMR, previously inaccessible by this method.     

Design,Synthesis, and Application of an Optimized Monofluorinated Aliphatic Label for Peptide Studies by Solid‐State 19F NMR Spectroscopy

A conformationally restricted monofluorinated α‐amino acid, (3‐fluorobicyclo[1.1.1]pentyl)glycine (F‐Bpg), was designed as a label for the structural analysis of membrane‐bound peptides by solid‐state ¹⁹F NMR spectroscopy. The compound was synthesized and validated as a ¹⁹F label for replacing natural aliphatic α‐amino acids. Calculations suggested that F‐Bpg is similar to Leu/Ile in terms of size and lipophilicity. The ¹⁹F NMR label was incorporated into the membrane‐active antimicrobial peptide PGLa and provided information on the structure of the peptide in a lipid bilayer.     

Prediction of folding stability and degradability of thede novo designed protein MB-1 in cow rumen

The authors have recently reported on the design of a protein (MB-1) enriched in methionine, threonine, lysine, and leucine. The protein is intended to be produced by rumen bacteria, in a way that would provide high producing lactating cows with limiting amino acids. In this report, MB-1 stability in the rumen is assessed, i.e., where the protein might be found after cell lysis or after being secreted by rumen bacteria. Current in vitro methods used to predict proteolytic degradability in the rumen were used for MB-1, as well as other natural proteins for comparison. MB-1 was found to be more susceptible to degradation than cytochrome c and ribonuclease A. Data indicate that MB-1 will be rapidly degraded if exposed to the rumen environment without protection. The contribution of folding stability to proteolytic stability was also examined. Rumen liquor components were selected to formulate a solution compatible with constraints of thermal denaturation studies. Denaturation curves show that the natural proteins were folded at rumen temperature. The MB-1 denaturation curves indicated that MB-1 does not unfold in a cooperative transition when heated from 20 to 70‡C. This suggests that MB-1 structure may be progressively modified as temperature increases, and that a continuum of conformations are available to MB-1. At 39‡C, a significant (50%) portion of MB-1 molecules had their tertiary structure unfolded, contributing to proteolytic degradability. Despite the unusual constraints used in MB-1 design (i.e., a maximized content in selected essential amino acids), results show that MB-1 has structural properties similar to previously reportedde novo designed proteins.     

Effects of chain length and N-methylation on a cation-pi interaction in a beta-hairpin peptide

The effects of N-methylation and chain length on a cation-pi interaction have been investigated within the context of a beta-hairpin peptide. Significant enhancement of the interaction and structural stabilization of the hairpin have been observed upon Lys methylation. Thermodynamic analysis indicates an increased entropic driving force for folding upon methylation of Lys residues. Comparison of lysine to analogues ornithine (Orn) and diaminobutyric acid (Dab) indicates that lysine provides the strongest cation-pi interaction and also provides the most stable beta-hairpin due to a combination of side chain-side chain interactions and beta-sheet propensities. These studies have significance for the recognition of methylated lysine in histone proteins.