Carbon Magnetic Resonance Studies of the Self-Aggregation of Calf Thymus Histones

Abstract

Self-aggregation of calf thymus histones H2A and H4 was studied by means of 13C magnetic resonance. Analyses of the changes in the intensities of several side-chain resonances, induced by added NaC1, confirm that carbon magnetic resonance can be used to monitor aggregation phenomena more accurately than other physicochemical methods. In particular the validity of an original computer-based method to treat 13C intensities is confirmed.The details of these aggregation phenomena are critically discussed in the light of a simple experiment on a I3C-enriched sample of H2B.Self-aggregation of histones, as induced by salt and/or pH changes has been thoroughly investigated during the last ten years by means of various physical techniques [l]. The recent progress in the knowledge of chromatin structure [2] has emphasized the importance of specific interhistone aggregations and thus, indirectly, also of the fairly specific self-aggregations presented by most histones. The detailed description of the mechanism of aggregation and of the secondary structure of the proteins in the aggregates has been given on the basis of a series of proton magnetic resonance (PMR) spectroscopy studies [3].I3C magnetic resonance is probably as efficacious in structural investigations ; this aspect is well illustrated by a considerable amount of data from recent literature, including a study [4] on H1, the only nonaggregating histone. However, as far as self-aggregation is concerned, carbon magnetic resonance spectroscopy is superior to PMR and to other commonly used physicochemical methods. In fact we have recently shown, in the case of H2B, that with the aid of a simple computer treatment of the 13C peak intensities it is possible to obtain an accurate description of the aggregate in a very direct and objective way This method is based on the peculiar variation of the intensities of many carbon resonances caused by self-aggregation. Under the only hypothesis that a single segment of the peptide chain is involved in aggregation, a systematic simulation of the intensity Abbreviations. CMR, carbon magnetic resonance; PMR, proton magnetic resonance; CD, circular dichroism. Single-letter code for amino acid residues: A (Ala), R (Arg), Q (Gln), E (Glu), G (Gly), I (Ile), L (Leu), K (Lys), T (Thr), V (Val).changes connected with rigid segments of various length and location along the sequence allows a clear identification of the rigid part of the chain.The present work extends this approach to histones H2A and H4, two of the other three aggregating histones. H3 was not studied by this simple method [6], since the presence of the oxidizable -SH groups may render the aggregation of this protein not comparable to that of H2B, H2A and H4.The limits of the automatic computer-based analysis were critically discussed with the aid of the data obtained on a sample of H2B specifically enriched on the methyl group of methionine. MATERIALS AND METHODSCalf thymus histones H2B, H2A and H4 were prepared using method I1 of Johns [7]. Purity tests were performed by...