Local Interactions Drive the Formation of Nonnative Structure in the Denatured State of Human α-Lactalbumin:  A High Resolution Structural Characterization of a Peptide Model in Aqueous Solution^,

Abstract

There are a small number of peptides derived from proteins that have a propensity to adopt structure in aqueous solution which is similar to the structure they possess in the parent protein. There are far fewer examples of protein fragments which adopt stable nonnative structures in isolation. Understanding how nonnative interactions are involved in protein folding is crucial to our understanding of the topic. Here we show that a small, 11 amino acid peptide corresponding to residues 101-111 of the protein alpha-lactalbumin is remarkably structured in isolation in aqueous solution. The peptide has been characterized by 1H NMR, and 170 ROE-derived constraints were used to calculate a structure. The calculations yielded a single, high-resolution structure for residues 101-107 that is nonnative in both the backbone and side-chain conformations. In the pH 6.5 crystal structure, residues 101-105 are in an irregular turn-like conformation and residues 106-111 form an alpha-helix. In the pH 4.2 crystal structure, residues 101-105 form an alpha-helix, and residues 106-111 form a loopike structure. Both of these structures are significantly different from the conformation adopted by our peptide. The structure in the peptide model is primarily the result of local side-chain interactions that force the backbone to adopt a nonnative 310/turn-like structure in residues 103-106. The structure in aqueous solution was compared to the structure in 30% trifluoroethanol (TFE), and clear differences were observed. In particular, one of the side-chain interactions, a hydrophobic cluster involving residues 101-105, is different in the two solvents and residues 107-111 are considerably more ordered in 30% TFE. The implications of the nonnative structure for the folding of alpha-lactalbumin is discussed.

Peptide and Protein Folding and Conformational Equilibria: Theoretical Treatment of Electrostatics and Hydrogen Bonding with Implicit Solvent Models

“…3a). It also demonstrates that modified radii lead to satisfactory agreement with the experimental results that residues 108-111 are largely unstructured (Demarest et al, 1999). Note that while such small adjustments of input radii can greatly improve the backbone interactions as reflected in better agreement of simulated conformational equilibria with experimental results, they do not significantly alter the overall electrostatic solvationfree energy.…”

“…1. The approach is then verified by folding simulations of a synthetic peptide with the sequence of (AAQAA) 3 and a small peptide from residues 101-111 of a-lactalbumin (a-lac) whose conformational equilibria have been determined previously (Demarest et al, 1999;Shalongo et al, 1994). We note that these efforts represent only preliminary steps in the development of a fully consistent implicit (GB) solvent force field, but the present studies illustrate the general approach one should use in developing such a force field.…”

Solution structure of a peptide model of a region important for the folding of ?-lactalbumin provides evidence for the formation of nonnative structure in the denatured state

“…This buffer was chosen to allow direct comparison with our studies of other peptide fragments of ␣LA. 5,6 The concentration of ␣lac:111-120 was determined from a molar absorption coefficient calculated using the method of Pace et al …”

“…These conditions were chosen to allow comparison with previous structural studies of other peptides derived from human ␣LA. 6 Experiments were carried out on Varian Unity INOVA 500 and 600 MHz spectrometers. Presaturation was used for solvent suppression.…”

“…[12][13][14][15][16] A more detailed description of this protocol is provided elsewhere. 6 For both the aqueous solution and 30% (vol/vol) TFE data, 100 structures were generated. Using a cutoff of 0.2 Å for upper-bound ROE violations and 5°for dihedral anglebound violations, 15 of the 100 structures determined in H 2 O were selected to represent the final acceptable ensemble for ␣lac:111-120 in aqueous solution.…”