R. Matthew Fesinmeyer

Hairpin folding rates reflect mutations within and remote from the turn region

Hairpins play a central role in numerous protein folding and misfolding scenarios. Prior studies of hairpin folding, many conducted with analogs of a sequence from the B1 domain of protein G, suggest that faster folding can be achieved only by optimizing the turn propensity of the reversing loop. Based on studies using dynamic NMR, the native GB1 sequence is a slow folding hairpin \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}(k_{{\mathrm{F}}}^{278}=1.5{\times}10^{4}/{\mathrm{s}})\end{equation*}\end{document}. GB1 hairpin analogs spanning a wide range of thermodynamic stabilities \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}({\Delta}G_{{\mathrm{U}}}^{298}=-3.09{\;}{\mathrm{to}}+3.25{\;}{\mathrm{kJ}}/{\mathrm{mol}})\end{equation*}\end{document} were examined. Fold-stabilizing changes in the reversing loop can act either by accelerating folding or retarding unfolding; we present examples of both types. The introduction of an attractive side-chain/side-chain Coulombic interaction at the chain termini further stabilizes this hairpin. The 1.9-fold increase in folding rate constant observed for this change at the chain termini implies that this Coulombic interaction contributes before or at the transition state. This observation is difficult to rationalize by “zipper” folding pathways that require native turn formation as the sole nucleating event; it also suggests that Coulombic interactions should be considered in the design of systems intended to probe the protein folding speed limit.

Possible locally driven folding pathways of TC5b, a 20-residue protein

A novel computational procedure for modeling possible locally driven folding pathways by stepwise elongations of the peptide chain was successfully applied to TC5b, a 20‐residue miniprotein. Systematic exploration of the possible locally driven pathways showed that the Trp‐cage structure of TC5b could be obtained by stepwise elongation starting from the noncentral local nucleation centers preexisting in the unfolded state of TC5b. The probable locally driven folding pathway starts with folding of α‐helical fragment 4‐9, followed by formation of the proper three‐dimensional structure of fragment 4‐12, and then 4‐18. Accordingly, the Trp‐cage‐forming interactions emerge successively, first Trp6–Pro12, then Trp6–Pro18, and then Trp6–Tyr3. The Trp‐cage‐like structures of TC5b found in this study by independent energy calculations are in excellent agreement with the NMR experimental data. The same procedure rationalizes the incomplete Trp‐cage formation observed for two analogs of TC5b. Generally, the success of this novel approach is encouraging and provides some justification for the use of computational simulations of locally driven protein folding. Proteins 2003;52:292–302.

Structural Insights for Designed Alanine-Rich Helices : Comparing NMR Helicity Measures and Conformational Ensembles from Molecular Dynamics Simulation

The temperature dependence of helical propensities for the peptides Ac‐ZGG‐(KAAAA)3X‐NH2 (Z = Y or G, X = A, K, and D‐Arg) were studied both experimentally and by MD simulations. Good agreement is observed in both the absolute helical propensities as well as relative helical content along the sequence; the global minimum on the calculated free energy landscape corresponds to a single α‐helical conformation running from K4 to A18 with some terminal fraying, particularly at the C‐terminus. Energy component analysis shows that the single helix state has favorable intramolecular electrostatic energy due to hydrogen bonds, and that less‐favorable two‐helix globular states have favorable solvation energy. The central lysine residues do not appear to increase helicity; however, both experimental and simulation studies show increasing helicity in the series X = Ala → Lys → D‐Arg. This C‐capping preference was also experimentally confirmed in Ac‐(KAAAA)3X‐GY‐NH2 and (KAAAA)3X‐GY‐NH2 sequences. The roles of the C‐capping groups, and of lysines throughout the sequence, in the MD‐derived ensembles are analyzed in detail.

Studies of helix fraying and solvation using 13C' isotopomers.

Both NMR and IR studies of carbonyl (13C′) isotopomers of designed helices can provide residue‐level details regarding the fractional occurrence and melting behavior of helical ϕ/ψ angles along the sequence of helical peptides, details that cannot be obtained from CD or 1H‐NMR studies. We have studied a classic series of helical models, Ac‐YGG‐(KAXAA)3K‐NH2 (X=A,V), in both aqueous and helix‐favoring media containing fluoroalcohol cosolvents, including a solvent system allowing the observation of cold denaturation. These studies confirmed the strong N‐capping associated with this sequence and revealed more extensive C‐terminal fraying than that calculated using current helicity prediction algorithms. In the X=A series, the central residues are somewhat resistant to thermal melting; it instead occurs predominantly at the frayable C terminus. For the X=V series under cold‐denaturing conditions, the temperature of maximal helicity is not uniform along the sequence and both solvated and nonsolvated helical alanine sites (13C=O stretches at 1592 cm−1 and 1615 cm−1, respectively) are apparent. Correlation between the two spectroscopies employed yielded the intriguing observation that the valine side chain is able to desolvate the i − 4 amide in short monomeric helices. In addition, we report further measurements of the temperature dependence of alanine statistical coil chemical shifts, the temperature dependence of the 13C chemical shift of urea (employed as chemical shift reference), and a useful formula for converting 13C′ shifts into fractional helicities.

Optimizing Aqueous Fold Stability for Short Polypeptides: 20 Residue Miniprotein Constructs That Melt as High as at 61 °C

There has been significant research activity directed at designing peptide sequences that are short but still display the folding cooperativity of proteins and the development of spectroscopic methods for assessing cooperativity [1]. Miniproteins as small 26-residues [2] and β-hairpin sequences that are relatively well-folded [3,4] have been reported. There was even a report of a 20 residue β-sheet, betanova [5]. Subsequent studies [6] indicate that betanova is less than 10% folded in water. As a result we adopted the production of 20 residue systems that are folded in water as a goal in our effort to define the features required for miniprotein stability. We have previously reported [7] that truncation and mutation of a sequence found in exendin-4 (a 39-residue peptide from Gila monster saliva) resulted in soluble 20-mer miniproteins that are fully (>90%) folded in water. Herein, we present the experiments that define the features required for fold stability and the extent of folding cooperativity displayed by these systems.