Peter D. Cary

Prothymosin α is a nuclear protein

The cellular location of the so‐called ‘thymic hormone’ prothymosin α has been studied by microinjection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that prothymosin α migrates to the nucleus at a rate comparable to that of histone H1. Prothymosin α cannot therefore be a hormone in the usual sense of the word.

The measurement of small nuclear overhauser effects in the 1H spectra of proteins, and their application to lysozyme

A simple differential technique for observing small Overhauser effects in 1 H spectra detected in the Fourier transform mode is described, equivalent to the homonuclear INDOR experiment in the continuous-wave mode. When the ring-current-shifted apolar resonances in lysozyme were irradiated using this technique, selective negative Overhauser effects were produced in the rest of the spectrum. This enabled other proton resonances in the same amino acid residues to be assigned. It also caused aromatic and exchangeable NH resonances to appear in the difference spectra, arising from the proximity of the apolar groups to the aromatic rings which cause the ring current perturbations. It is shown how these effects aid assignment in a protein of known structure, and can in principle give structural information about a protein of unknown structure.

Comparison of the structures of the chromosomal high mobility group proteins HMG1 and HMG2 prepared under conditions of neutral and acidic pH

The chromosomal proteins HMG1 and 2 have been prepared by salt extraction and phosphocellulose chromatography at neutral pH (Isackson, P.J., Debold, W.A. and Reeck, G.R. (1980) FEBS Lett. 119, 337-342) to minimize protein denaturation. The structures of these phosphocellulose-prepared high mobility group proteins have been compared with those of high mobility group proteins using the previously described acid-extraction conditions which fully denature the proteins. When compared in the same solvent conditions the acid-extracted proteins did not refold to give the same level of alpha-helical and tertiary folded structures as the phosphocellulose-prepared proteins, suggesting that acid treatment can cause some irreversible damage to the proteins. This finding was supported by changes in the structure observed when phosphocellulose-prepared HMG1 was neutralized after exposure to acid. Gel filtration studies reveal no differences in the size of the high mobility group proteins, phosphocellulose-prepared and acid-extracted proteins both being largely monomeric in solution. Little difference was detected in the DNA-binding properties of the two types of protein, nor was there any difference in the oxidation state of the cysteines. However, isoelectric focusing analysis revealed differences in the subfractions of HMG2 prepared by the two methods.

Histone H1 structure probed by Staphylococcus aureus V8-proteinase.

Proteolytic digestion of calf thymus histone H1 with Staphylococcus aureus V8-proteinase under structuring conditions generates one major limit peptide P1 which consists of approx. 170 residues. Edman degradation establishes the N-terminal sequence as: Leu-Ile-Thr-Lys-Ala-Val-Ala-Ala-Ser-Lys. Chymotryptic fingerprinting shows that the C-terminal part of the H1 molecule is fully preserved. The peptide therefore comprises the residues H1 (42-210). The Glu-41 cleavage is extremely unusual as it occurs in the structured G-domain which is known to be resistant to proteinases (Hartman, P. G., Chapman, G. E., Moss, T. and Bradbury, E. M. (1977) Eur. J. Biochem. 77, 45-71; Böhm, L., Sautière, P., Cary, P. D. and Crane-Robinson, C. (1982) Biochem. J. 203, 577-582). The V8-proteinase cleavage product H1 (42-210) shows only 20% folding as compared to 95-99% folding shown by the peptides H1 (34-121), H1 (31-210) and H1 (33-210). Folding of the G-domain thus critically depends upon the presence of the eight residues 33-41 amongst which the Gly-Pro-Pro sequence at position 36-38 and a beta-turn predicted at position 35 are considered to be particularly important. The location of the cleavage site in the G-domain renders Staphylococcus aureus V8-proteinase suitable as a structural probe.

Preparation of chromosomal protein A24 (uH2A) by denaturing gel filtration and preparation of its free nonhistone component ubiquitin by ion-exchange chromatography

Chromosomal protein A24 (uH2A) is unique in that it comprises the nucleosomal core histone H2A in isopeptide linkage with the highly conserved, globular, and stable nonhistone protein ubiquitin. Some 10% of the chromatin complement of H2A is modified in this way and studies to elucidate a role for this modification have concentrated on observations requiring no purification of A24 due to the difficulty in isolating the protein in large and pure quantities. We describe a method for isolating A24 by chromatography on Pharmacia G-100 gel filtration medium under urea denaturing conditions. A24 prepared by this method is structurally intact and is available in the quantities required for studies of the behavior and influence of the protein on histone-histone, histone-DNA, and enzymatic interactions. In conjunction with this method we describe a procedure for the isolation of large quantities of free ubiquitin of far greater purity than previously reported.