Thermal Stability Comparison of Purified Empty and Peptide-Filled Forms of a Class I MHC Molecule

Abstract

A secreted form of a class I major histocompatibility complex (MHC) molecule was denatured and renatured in vitro in the absence of peptide. The resulting empty class I heterodimer was immunologically reactive and structurally similar to a heterodimer renatured in the presence of an appropriate restricted peptide. Thermal stability profiles indicated that the two forms of heterodimer differed in their resistance to denaturation by heat but that a significant portion of the empty class I heterodimers had a native conformation at physiological temperatures. Free energies calculated from these data gave a direct measure of the stabilization of the class I MHC molecule that resulted from peptide binding.

Peptide-Independent Folding and CD8αα Binding by the Nonclassical Class I Molecule, Thymic Leukemia Antigen

“…The T m value of 54°C is similar to the values (54 and 57°C) reported for H-2K d bound to specific peptide ligands (35,38). Most class Ia molecules, like HLA-A2 in the present study, fail to assemble in the absence of appropriate peptides, indicating that peptide is an integral component of the properly folded molecules.…”

“…A second transition with T m Ͼ60°C characterized by a sign reversal of the CD signal probably represents unfolding of free ␤ 2 m (35,40). The 54°C T m of empty T18 d is similar to that reported for H-2K d peptide complexes (54 -57°C) (35,38) and greater than that reported for the class Ib protein T10 (49°C) (40), which does not bind peptides. Thus empty T18 d molecules are relatively stable and folding the protein in the presence of peptide does not increase its stability.…”

“…H-2K d is unusual in that it can be generated as an empty molecule. However, the T m of empty H-2K d (45°C) (35) is considerably lower than the value for peptide-free TL. Thermal denaturation studies have been performed with one additional nonclassical class I molecule, T10 (40).…”

“…The subsequent spectra taken at 25°C are similar to those obtained with unheated samples, demonstrating that thermal denaturation is largely reversible under the conditions used. Thermal denaturation profiles demonstrated a T m of 54°C, reflecting dissociation and unfolding of the H chain (35). The T m is identical for T18 d folded in the presence or absence of the nonameric peptide library.…”

“…Far-UV CD spectroscopy has been used to analyze the structure and thermal stability of a variety of classical and nonclassical MHC molecules (17,(35)(36)(37)(38)(39)(40). The far-UV spectra of T18 d are shown in Fig.…”