Thermodynamics of protein cross-links

Abstract

The thermal transitions of native lysozyme and a well-characterized cross-linked derivative of lysozyme [Imoto, T., and Rupley, J. A. (1973), J. Mol. Biol. 80, 657] have been studied in 1.94 M guanidine hydrochloride at pH 2. The observed increase in the melting temperature from 32.4 degrees C for native lysozyme to 61.8 degrees C for the cross-linked derivative corresponds to a calculated 5.2 kcal/mol increase in the free energy of denaturation. This free-energy change is attributed to the decreased entropy of the unfolded polypeptide chain following introduction of a cross-link and is shown to compare well with theoretical predictions. The possibility that an introduction of a cross-link could also affect the enthalpy of an unfolded protein was investigated. The heats of reduction of bovine serum albumin and lysozyme by dithioerythritol in 6 M guanidine hydrochloride were determined and compared to that for the model peptide, oxidized glutathione. The near identity of the observed heats was taken as evidence that the introduction of cross-links into a random-coil protein does not, in general, introduce strain.

A three‐disulphide intermediate in refolding of reduced ribonuclease a with a folded conformation

“…Assuming the stability to be related linearly to the urea concentration [23], the conformational stability of IIIn in the absence of urea is likely to be only half the 10.6 kcal/mol estimated for native RNase [24]. This decrease in stability of 5 kcal/mol is about that expected simply for the absence of 1 covalent crosslink [25].…”

A Protein’s Conformational Stability Is an Immunologically Dominant Factor: Evidence That Free-Energy Barriers for Protein Unfolding Limit the Immunogenicity of Foreign Proteins

“…It has been reported that HEL was stabilized by cross-linking between K1 and H15 with an alkyl chain (1-15 CL-HEL) (29) and by cross-linking between E35 and W108 through an ester bond (35-108 CL-HEL) (21,30). These modified amino acids are shown in the three-dimensional structure of HEL (Fig.…”

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

“…RNase Thl was found less stable to heat than the intact protein (-ATm 16"C), whereas RNase Th2 was fully unfolded at room temperature. The peptide bond hydrolysis is expected to destabilize a protein for purely entropic reasons, analogous to the reductive cleavage of a disulfide bond in a protein (Johnson et al, 1978;Pace et al, 1988). In fact, the destabilization due to fission of a covalent bond is related to the entropy gain in the denatured state, even if enthalpy and heat capacity effects may be included (see Krokoszynska & Otlewski, 1996, for a recent discussion).…”