William Shalongo

Dichroic statistical model for prediction and analysis of peptide helicity.

Traditional statistical models for the prediction of peptide helicity are written in terms of the mean fractional helicity of the peptide residues. Far ultraviolet circular dichroic measurements of peptide solutions are converted to mean fractional helicity by partitioning the observed ellipticity between that of a perfect helix and a random coil. This partition does not adequately represent the ensemble of peptide molecules present in solution that populate imperfect helical conformations of quite variable lengths. A new dichroic statistical model has been written in terms of ellipticity rather than fractional helical content that recognizes (1) the source of ellipticity, peptide bond adsorption; (2) the differential ellipticity of peptide bonds in the terminal and interior helical turns; and (3) the contributions of each participant in a conformational ensemble to the observed ellipticity. Comparative analyses of host/guest peptides indicates that significant differences are obtained between residue w and n weights and ellipticity values using the traditional and dichroic statistical models. Proteins 28:467–480, 1997.

Analysis of N‐terminal capping using carbonyl‐carbon chemical shift measurements

The model peptide XAAAAEAAARAAAARamide is used to examine the contributions of an N‐terminal capping interaction to the conformation and stability of a helical ensemble. The reference peptide has an alanine residue at position X while the capping peptide has a serine residue at this position. The helical ensemble was characterized using circular dichroism measurements and carbonyl‐carbon chemical shift measurements of selectively enriched residues. The distribution of helicity within the ensemble of the reference peptide at pH 11 and 0°C appears symmetrical, having a uniform central helix and frayed ends. This distribution is truncated at pH 6 by the repulsive electrostatic interaction between the positively charged α‐amino group and the positively charged end of the helical macrodipole. The capping peptide forms a side‐chain/main‐chain hydrogen bond involving the serine residue and amide of alanine 4. The presence of this hydrogen bond generates a unique motif in the chemical shift profile of its helical ensemble. The conformational stabilization contributed by this hydrogen bond, although cooperatively distributed throughout the helical ensemble, is preferentially focused within the first helical turn. The stabilization provided by this hydrogen bond is able to offset the truncation of the helical ensemble generated by the repulsive electrostatic interaction observed at pH 6. Proteins 33:167–176, 1998.

Evidence for glutamate self-capping within a peptide helix.

The thermal dependence of the carbonyl carbon chemical shift of each residue in a helical peptide may be analyzed in terms of a two-state helix/coil transition. Such analyses generate values for the chemical shift of each residue in the helical and in the coil conformational ensembles of the peptide. The sequence dependence of the difference in these two values, termed the difference chemical shift, provides a description of the mean distribution of helicity within the helical ensemble. In this report, we improve two aspects of the procedures used to analyze prior chemical shift measurements of the helical peptide acetylW (EAAAR)3Aamide. The new difference chemical shift values for 16 of the 18 residues describe a very uniform central helical ensemble with frayed ends. However, the difference chemical shift values for the two remaining residues, alanines 03 and 08, are significantly diminished relative to this uniform distribution. Each of these two alanine residues is located i-4 to a glutamate residue. It is suggested that the difference chemical shifts for these two alanine residues are diminished by a self-capping interaction within the i + 4 glutamate residues.

Model Simulations of the Kinetics of Protein Folding

Publisher Summary The protein-folding problem—the prediction of three-dimensional structure from sequence—continues as a major focus of protein research. Experimentalists observe the kinetics of protein folding in order to understand the sequence of events which directs folding into a unique structure. An experimental signal is selected whose mean amplitude changes during the folding process. Folding is initiated by rapid injection of a solution of unfolded protein, frequently in excess denaturant, into a solvent in which the folded protein is more stable. This chapter discusses the kinetics of model-folding mechanisms of increasing complexity. In the chapter, the simulated kinetics are subjected to traditional exponential analysis. The chapter presents exponential analysis as providing meaningful descriptions of the component reactions in protein folding only under very limited conditions and discusses analysis by mechanism simulation as a more promising alternative. Protein folding is represented by a series of coupled reversible isomerization reactions, each exhibiting first-order kinetics. Such representations exclude second-order reactions involved in the formation of stable quaternary structures, nonspecific aggregates, and chaperonin complexes.

Simulation of Protein Hydrodynamic Changes Observed by Urea Gradient Gel Electrophoresis

The kinetics of protein conformational changes are normally monitored by absorbance or fluorescence measurements since spectral techniques are amenable to data collection on a rapid time scale. Unfortunately, these spectral techniques often reflect local changes in tertiary structure and cannot be assumed to reflect the initial hydro-dynamic collapse of a randomly coiled denatured polypeptide into a compact structure. Since such a collapse is likely an early event in protein folding, it would be useful to have a procedure available to observe the kinetics of the hydrodynamic change accompanying protein folding and unfolding. Creighton (1979, 1980) lias demonstrated that zone electrophoresis in polyacrylamide slab gels containing a urea gradient can provide information regarding the kinetics of the hydro-dynamic changes accompanying unfolding or refolding in urea, the presence of multiple forms of the protein, and the transient accumulation of intermediate forms. Unfortunately, the protein profiles observed upon staining of urea gradient gels after electrophoresis can only be qualitatively interpreted. While Creighton has employed the equation of Mitchell (1976) to simulate such profiles, we find that this equation is awkward to use and can give unreliable results. In this report we illustrate the application of the equation of Endo et al. (1983) to such simulations.