Conformation and unfolding thermodynamics of epidermal growth factor and derivatives

Abstract

Mouse submaxillary epidermal growth factor (EGF) is a 53-residue single chain peptide hormone of known amino acid sequence which contains three disulfides, five tyrosines, and two tryptophans. Circular dichroic (CD) spectra have been obtained and resolved for EGF, several well-characterized chemical and enzymic derivatives, and related low molecular weight model compounds. Assignments have been made to most of the resolved bands; these include the peptide, aromatic, and disulfide chromophores. From a comparison of the rotational strength of the 213-nm resolved CD band in native EGF with that of standard proteins, EGF is estimated to contain about 22% beta structure and no alpha helicity. A derivative of EGF lacking the five carboxyl-terminal residues (prepared by limited trypsin digestion) and the cyanogen bromide derivative, in which there is a single main-chain cleavage at residue 21, have spectra properties indicative of approximately 10 and 12% beta structure, respectively. The near-ultraviolet CD spectra of the derivatives are similar to, albeit not identical with, that of EGF. The rotational strengths characteristic of the side-chain chromophores in EGF and these derivatives are several-fold higher than the corresponding values in low molecular weight model compounds. Thus, it appears that EGF and these modified forms contain a stable (and similar) tertiary structure. In contrast, the S-aminoethylated derivative of EGF exhibits a drastically altered CD spectrum relative to EGF indicating a different conformation(s). Equilibrium studies on the guanidinium hydrochloride (GdmCl) mediated reversible unfolding of EGF showed that the transition midpoint is quite high (i.e., 6.89 M GdmCl at 25.0 degrees C), thus, indicating considerable stability. From these data a rough estimate of 16 kcal/mol can be made for the unfolding free energy (delta G degrees) of EGF in the absence of denaturant. Interestingly, EGF exhibits greater stability characteristics than several proteins two to four times its size. The cyanogen bromide derivative of EGF exhibited greatly reduced stability characteristics, e.g., the transition midpoint occurred at 4.19 M GdmCl (25.0 degrees C) and delta G degrees was estimated to be approximately 4 kcal/mol. Thus, a single main-chain cleavage reduced the stability of EGF by about 70%. Thermal transitions of EGF and the cyanogen bromide derivative in the presence of concentrated GdmCl are characterized by a relatively high enthalpy of about 25 kcal/mol at 40 degrees C and a low (probably zero) heat capacity. From these thermodynamic parameters one can calculate that the large reduction in delta G degrees due to scission of the single peptide bond between residues 21 and 22 can be attributed almost completely to a change in entropy; e.g., at 40 degrees C the apparent entropy of unfolding of EGF is 20.4 cal mol-1 deg-1 while that of the cyanogen bromide derivative is 66.4 cal mol-1 deg-1.

A high resolution 1H NMR study of the solution structure of human epidermal growth factor

“…It is interesting to note that one residue from each disulphide bridge is located in this /3-80 sheet region. The location of the &sheet is in good agreement with the structure prediction of Holladay et al [25] for murine EGF, although there are important differences; residues 34-37, for example, were predicted to be in a sheet while we find this region is involved in a type II turn. A complete determination of the solution structure of human EGF using distance constraints from NMR is now required.…”

Rat epidermal growth factor: complete amino acid sequence

“…In contrast, it has been suggested by Komorya et al [41] that the primary functional role of the amino and carboxyl-terminal regions of mEGF is to provide conformational stability for the middle region of the molecule (residues 20-31) which these workers claim as the receptor binding region. Further support for this proposal is provided by the reduced B-sheet conformation reported for mEGF-(1 -47) [42]. The equivalent potency of rEGF (all four forms) and mEGF demonstrated in this study suggests that the conformation of rEGF (which lacks 1-3 amino and 5 carboxy1-terminal residues) and mEGF are comparable.…”

Effects of Different CMV-Heat-Inactivation-Methods on Growth Factors in Human Breast Milk

“…The high stability of EGF is reported to be based on its stable tertiary structure (13). Molecular stability is shown not only in terms of pasteurization techniques (14), but also in disulfide scrambling techniques using urea or guanidine chloride (15).…”