Apolar Residues Research Articles

Determination of the hydrophobic binding site of phosphatidylcholine exchange protein with photosensitive phosphatidylcholine.

1-Acyl-2-(7-(4-azido-2-nitrophenoxy)-[1-14C]heptanoly)-sn-glycero-3-phosphocholine was synthesized in order to study the lipid-binding site of the phosphatidylcholine exchange protein from bovine liver. Photosensitive phosphatidylcholine was incorporated into the protein by incubation with vesicles of this phosphatidylcholine derivative. The lipid-protein complex was separated from the vesicles by chromatography on Biogel A-0.5m. Photolysis of the complex by irradiation with light of a high pressure mercury lamp at a wavelength above 340 nm generated the highly reactive nitrene. Sodium dodecyl sulfate gel electrophoresis of the photolysed complex indicated that 30% of the endogenous 14C-labeled phosphatidylcholine was covalently linked to the protein. Peptides were isolated after digestion of the photolysed complex with protease from Staphylococcus aureus and trypsin. It was determined that the 2-acyl chain of the phosphatidylcholine molecule was linked to the peptide segment -Gly-Ser-Lys-Val-Phe-Met-Tyr-Tyr-. This segment was part of a protease peptide of about 65 residues of which the sequence was determined by Edman degradation for the first 38 residues. This peptide contains a cluster of apolar residues -Val-Phe-Met-Tyr-Tyr-Phe with an extremely high hydrophobicity index and with a predicted beta-sheet conformation. It is concluded that this hydrophobic cluster forms part of the binding site.

Hydrodynamics and protein hydration

We have critically evaluated hydrodynamic data from 21 proteins whose molecular dimensions are known from X-ray crystallography. We present two useful equations relating the molecular weights and sedimentation coefficients of globular proteins. The hydrodynamic data combined with data for small molecules from the literature indicate that failure of the Stokes equation occurs only for molecular weights <850. Calculated hydration values for the 21 proteins have a mean value and standard deviation of 0.53 ± 0.26 g H 2O/g protein. Furthermore, statistical arguments indicate that only 5.3% of the variance is due to experimental error. The mean value and especially the dispersion of values are in sharp contrast to the values 0.36 ± 0.04 obtained by others from nmr measurements on frozen protein solutions. Hydration values calculated from nmr measurements are closely correlated with the number of charged and polar amino acid residues. In contrast to this result, our analysis of the amino acid compositions of the four proteins with the lowest hydration and the four monomeric proteins with the highest shows that the range of values we observe cannot be accounted for on the basis of amino acid composition. In fact there appears to be a weak correlation between the number of apolar residues and hydrodynamic hydration. We therefore conclude that the dispersion must result from variations in fine details of the surface structures of individual proteins. We propose a model of hemispherical clathrate cages which if correct, would account for the differences in the data obtained by these two methods.

Resistance of lipophilin, a hydrophobic myelin protein, to denaturation by urea and guanidinium salts.

The influence of urea, guanidinium chloride (GdmCl), and granidinium thiocyanate (GdmSCN) on the solution structure of lipophilin was examined by circular dischroism and fluorescence techniques. According to the CD results, lipophilin retained at least 60% of organized secondary structure in 8 M urea and 6 M GdmCl (measurements were not possible in GdmSCN). This partial denaturation was of a complex, irreversible nature, and was not appreciably enhanced by prolonged incubation (8 days), by heating to 70 degrees C, by disulfide bond reduction, or by pH variation in the range pH 1.5 to 11. Fluorescence studies demonstrated that the tryptophan residues were only slightly perturbed by 8 M and 6 M GdmCl and remained well buried to the permeant quenching agent acrylamide. A greater, but still far from complete, disruption of lipophilin was achieved in 6 M GdmSCN, and fluorescence polarization provided evidence for some form of cooperative structural change induced by increasing concentrations of this reagent. Transfer of the protein from 2-chloroethanol, in which the tryptophan residues are fully exposed, into 6 M GdmSCN by dialysis resulted in reburial of the fluorophores owing to development of tertiary structure. The combined evidence suggests that the extraordinary resistance of lipophilin to these denaturants is due to the presence of an impervious hydrophobic core. In lipophilin and some other membrane-associated proteins, extended sequences of apolar residues might provide the nuclei for such structural domains.

A nuclear-magnetic-resonance study of the globular structure of the H1 histone.

The globular structure of the H1 histone has been studied by 1H NMR, as an aid to elucidating its mode of action, and also as a model small protein molecule for investigating the capabilities of NMR for gaining information about the tertiary folding when the structure has not been determined by other techniques. Guanidylation of the lysine residues does not effect the capability of the peptide chain to fold into the native globular structure, suggesting that the lysine residues are on the surface of the structure. Partial assignment has been made of some of the ring‐current‐shifted resonances (i.e. resonances from methyl groups of apolar residues which are shifted by their proximity to the magnetic fields of aromatic rings) by a combination of spin‐decoupling experiments and theoretical ring‐current‐shift stimulations. Substitution of the tyrosine residue of histone H1 considerably weakens the globular structure, and observation of specific changes in the perturbed methyl region of the spectra on substitution indicates the aromatic residues giving rise to ring‐current perturbation of specific peaks in the spectrum. Interproton Overhauser effects are demonstrated, arising from throughspace dipole interaction between protons of the aromatic residues and methyl residues, the resonances of which are perturbed by close proximity to the aromatic rings. The results of these experiments confirm the assignments made from the results of the tyrosine modification experiments.

Fibrinogen: A preliminary analysis of the amino acid sequences of the portions of the α, β and γ-chains postulated to form the interdomainal link between globular regions of the molecule

It has been suggested (Doolittle et al., 1977) that portions of the α-, β- and γ-chains of fibrinogen form a coiled-coil rope of α-helices and that this rope connects globular domains of the molecule. A fast Fourier transform analysis of the relevant amino acid sequences has shown that there is a significant 3.5-residue period in the linear disposition of the apolar residues in all three chains. This periodicity is characteristic of amino acid sequences of α-fibrous proteins, such as α-tropomyosin and α-keratin, where the tertiary structure is closely related to a coiled-coil of α-helices. However, a detailed study of the fibrinogen sequences shows that the structure is likely to contain several regions which do not have a simple secondary structure. The detailed conformation of the postulated rodlike region of fibrinogen is therefore complex and may approximate a coiled-coil only over relatively short lengths. An important question to emerge from this analysis is whether correct positioning of apolar residues in a pseudo-repeating heptad is sufficiently important to override low α-helix-favouring potential of other residues in the heptad.

Enthalpy of interaction between some cationic polypeptides and n-alkyl sulphates in aqueous solution

The enthalpies of interaction of a homologous series of n-alkyl sulphates with poly(L-lysine)-hydrobromide, poly(L-arginine)hydrochloride and poly(L-histidine)hydrochloride have been measured at 25°C. A linear relationship between the enthalpy of interaction and carbon chain length has been found for alkyl chain lengths above C8. The data support a model based on a stoichiometric interaction between the anionic head group of the n-alkyl sulphates and the cationic side chains of the polypeptides. The results are discussed in relation to the interaction between surfactants and proteins, and lead to the view that a major contribution to the enthalpy of interaction arises from the surfactant–cationic residue interactions, but that there remains an additional contribution from surfactant–apolar aminoacid residue interactions.

Chiral solvent-induced asymmetric synthesis. Part 2. Photosynthesis of optically enriched hexahelicenes

Irradiation of 2-styrylbenzo[c]phenanthrene (1) in eleven different, chiral solvents gave rise to the formation of non-racemic hexahelicene with optical yields of 0.2–2.0%. The role of the chiral solvents is ascribed to their influence on the equilibrium between enantiomeric conformations of cis-(1), which leads to an excess of one of them. The magnitude and sign of the effect can be related to the size and position of large apolar residues at the chiral centre in the solvent molecules. Similar experiments were done with some other precursors of hexahelicene and some of its derivatives. It is concluded that the solvent behaves as a matrix, in which the photoreaction takes place.

Solvation and conformational effects in aqueous solutions of biopolymer analogues.

Biopolymer conformational transitions play a fundamental role in life processes. These transitions are triggered and controlled by subtle changes in the ‘solvent medium’ and are therefore likely to depend on the hydration states of the biopolymer and the soluble small molecule species involved. Three distinct types of hydration behaviour common to biopolymer systems are discussed: (1) Ionic hydration is of an electrostatic nature, i.e. long range and radial but specific effects exist in the manner in which water molecules are oriented about different ions and this may well be the origin of the various lyotropic series of ion specific effects which are so common in colloid, polymer and biochemistry. (2) The term ‘specific hydration’ is used to describe the direct interaction by hydrogen bonding between water and polar sites on organic molecules capable of acting as proton donors or acceptors. Since the hydrogen bond potential is very orientation specific, it follows that ‘specific hydration’ effects depend sensitively on the detailed stereochemistry of the molecular hydration sites, i.e. their distances of separation and mutual orientations. Thus in cases where molecules can exist in several anomeric or diastereoisomeric forms, or where different conformational states can arise by rotation about carbon-carbon bonds, specific hydration interactions may significantly affect the conformational energy minimum such that solvent dependent conformational states may exist. Similarly the solvent will influence the positions of the equilibria between various isomeric states, e.g. of sugars. (3) Hydrophobic hydration probably arises from the reorientation of water molecules in the vicinity of an apolar molecule or residue, such that the OH vectors are not directed towards the apolar moiety. This is an entropically unfavourable process which can be partly reversed by the association of two (or more) such hydrophobically hydrated residues. In this way some of the perturbed water can relax back to its normal bulk state in which more molecular orientations are possible. A reassessment of the hydrophobic interaction shows, however, that contrary to currently held views, the potential well due to two alkyl groups in aqueous solution is shallower than it is for two similar groupsin vacuo, and also that the entropy gain from the pair interaction is not as large as has been believed. These findings necessitate a reappraisal of the molecular details of the hydrophobic interaction.

Structural requirements for steroid binding and quenching of albumin fluorescence in bovine plasma albumin

1. 1. Steroids interact with bovine plasma albumin at a binding region that involves tryptophanyl, tyrosyl, arginyl and lysyl residues. The function of the tryptophanyl residues is demonstrated by: (a) the decrease of albumin binding affinity after modification of one tryptophanyl with 2-nitrophenylsulfenyl chloride; (b) steroid quenching of albumin tryptophanyl fluorescence; and (c) steroid quenching of 1-anilinonaphthalene-8-sulfonate fluorescence, when it is excited by energy transfer from excited tryptophanyls. The function of tyrosyl residues is demonstrated by the decrease of albumin binding affinity after nitration of 30% tyrosyls with tetranitromethane, or deprotonation of tyrosyls by variation of pH. The function of arginyl and lysyl residues is demonstrated by the decrease of binding affinity after modification of these residues with glyoxal, formaldehyde or acetic anhydride. The presence of both apolar (Trp, Tyr and Lys (deprotonated))and polar(Arg and Lys(protonated)) residues at the steroid binding site fits in well with the site relative apolarity, when expressed on the Kosower scale (Kosower, E.M. (1958) J. Am. Chem. Soc. 80, 3253–3260). 2. 2. The contribution of specific amino acid residues to steroid binding depends to some extent on the steroid structure, as exemplified by the quantitatively different role of arginyl (or lysyl) residues in albumin interaction with testosterone acetate and epitestosterone, respectively, or that of tyrosyl residues in albumin interaction with 11-deoxycorticosterone and epitestosterone, respectively. 3. 3. The concerted action of polar and apolar amino acid residues is an essential requirement for steroid binding, since unfolding of albumin polypeptide chain by guanidine-HCl, urea, or by reduction of disulfide bridges with 2-mercaptoethanol,

Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea.

The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 degrees C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not beta-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5-9.5 at 30 degrees C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 degrees C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/mo, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized.

Apoprotein stability and lipid-protein interactions in human plasma high density lipoproteins.

Temperature-dependent conformational changes of the principal apoprotein of human plasma high density lipoprotein (HDL), apoA-I, have been studied in the isolated apoprotein, in complexes of apoprotein with phospholipid, and in intact HDL. Differential scanning calorimetry shows that in solution apoA-I undergoes a reversible, two-state thermal denaturation (midpoint temperature 54 degrees). The enthalpy (2.4 cal/g)(10.0 J/g) and specific heat change (0.08 cal/degrees C per g)(0.33 J/degrees C per g) associated with the denaturation were used to calculate the free energy difference (deltaG) between native and unfolded apoA-I at 37 degrees. DeltaG (2.4 kcal/mol)(10.0 kJ/mol) is less than that of other globular proteins (typically 8-14 kcal/mol)(33-59 kJ/mol), indicating that at 37 degrees native apoA-I has a loosely folded conformation. Turbidity studies show that apoA-I is able to solubilize phospholipid in its native but not in its denatured form. Mixtures of apo-HDL (the total apoprotein of HDL) or apoA-I with dimyristoyl lecithin show a thermal transition at about 85 degrees not present in the lecithin or the apoprotein alone, which indicates that the native conformation of the apoprotein is stabilized by phospholipid. Scanning calorimetry of intact HDL shows a high-temperature endotherm associated with disruption of the HDL particle, suggesting that in HDL the conformation of apoA-I is also stabilized by interaction with lipid. The loosely folded conformation of native, uncomplexed apoA-I may be especially adapted to the binding of lipid, since this process may involve both hydrophobic sites on the surface of the protein and concealed apolar amino acid residues that are exposed by a cooperative, low energy unfolding process.

Isolation and characterization of band 3, the predominant polypeptide of the human erythrocyte membrane

Band 3 is the predominant approximately 90,000-dalton polypeptide component of the human erythrocyte membrane. It was solubilized selectively, along with the other major glycoproteins, by extracting membrane ghosts with Triton X-100 under nondenaturing conditions. Two major polypeptides remained associated with Band 3 under these conditions; however one (Band 6) could be dissociated at an ionic strength of 0.15 and the other (Band 4.2) by treatment with p-chloromercuribenzoate. Band 3 was then purified (greater than or equal to 97%) by aminoethyl cellulose ion exchange chromatography. The isolated protein was free of phospholipid and was moderately enriched in apolar amino acid residues; it contained galactose and glucosamine but very little sialic acid and galactosamine. When Band 3 was labeled by treatment of ghosts with galactose oxidase plus KB3H4 and then purified, the electrophoretic mobility of its radioactivity lagged slightly behing that of its Coomassie blue staining profile. Variation in glycosylation could therefore cause the diffuse trailing zone characteristically observed for Band 3 on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The ultraviolet circular dichroism of Band 3 was stable in nonionic detergent and suggested an alpha helix content of 43%, a value close to that estimated for this polypeptide in the membrane.

Incorporation of ( 3H)-arachidonic acid into cattle retina lipids: High uptake in triacylglycerols, diacylglycerols, phosphatidylcholine and phosphatidylinositol

The incubation of ( 3H)-arachidonic acid-prelabeled cattle retinas for 20 min in the presence of glucose under a gas phase of 5% carbon dioxide in oxygen showed uneven labeling in lipid classes. Total phospholipids, acylglycerides and free fatty acids contained 35, 37 and 31 per cent of the total radioactivity. In phosphatidylinositol and phosphatidylcholine almost 70% of the polar lipid ( 3H)-arachidonate was recovered. About 70% of the total fatty acid esterified in retina lipids was found in diacylglycerols, triacylglycerols, phosphatidylinositol and phosphatidylcholine. It is concluded that the cattle retina “in vitro” takes up free arachidonic acid and that this fatty acid is further unevenly acylated into lipids. The apolar fatty acyl residues of lipids display an independent turnover and their composition may be modified by acylation-deacylation reactions. In several cellular lipids, a differential turnover of the fatty acids as compared with other lipid moieties has been indicated, such as the case of phosphatidylinositol (1–3) and cardiolipin (4). The latter is enriched in the inner mitochondrial membrane where energy conservation processes take place and the former has been implicated in synaptic transmission (5) and related with a protein identified as the acetylcholine receptor (6). In brain phosphoinositides tetraenoic molecular species are by far the largest (2) and an active acylation-deacylation cycle of arachidonic acid occurs (7). However data regarding retina phosphoinositides composition and metabolism is limited to: fatty acid distribution (8), to some studies on the phosphodiester metabolism by 32p (9) and to a study reporting that in frog rod outer segments and retina, polyphosphoinositides are undetectable (10). The purpose of the present investigation was to observe the ( 3H)-arachidonic acid labeling of acylglycerides and of phosphoglyceride classes of cattle retina.

Analysis of the primary sequence of alpha-tropomyosin from rabbit skeletal muscle.

The amino acid sequence of α-tropomyosin, the main component of rabbit skeletal tropomyosin, has been completed by Stone et al. (1974) . The sequence has been analysed computationally and some regularities noted. A periodicity of about 20 residues has been confirmed in the linear distribution of both the acidic residues and also the apolar residues not confined to positions 2 and 6 in the pseudo repeating heptapeptides characteristic of tropomyosin. Glutamic acid and also lysine and arginine are found in significant quantities in positions 3 and 5, respectively. Using the conformational parameters listed by Chou & Fasman (1974) , it is shown that tropomyosin is most probably completely α-helical with any possible non-helical regions confined to the N- and/or C-termini. Short portions of the amino sequence low in α-helix-favouring residues show a well defined 40-residue period. The 40- and 20-residue periods may be correlated directly to the actin separation along one strand of the thin filaments of skeletal muscle. As recent experimental evidence by Johnson & Smillie (1975) and Stewart (1975) shows that the two chains of tropomyosin are in axial register, the nature of the packing of apolar residues between chains has been reassessed. The brush end of the Mg tactoids is shown to correspond to the N-termini of the tropomyosin molecules. Troponin is therefore located close to the part of the chain sequence containing the single cysteine residue. It is also shown that the Mg tactoid is produced by divalent Mg cations bridging acidic residues of oppositely directed molecules. The effective repeat length of tropomyosin in the thin filament corresponds to 277±5 residues.

A review of statistical structures in synthetic polypeptides and biological macromolecules

The relationship between the structure and function of biological macromolecules is fundamental to our understanding of molecular biology. It is generally true that those proteins which are rich in charged residues have a high axial ratio (fibrous proteins) and those which are low in charged residues tend to have a small axial ratio (globular proteins). Rod-shaped proteins have a larger surface area and smaller internal volume per unit volume than do the globular proteins. The surface charge per unit area is reduced by the protein adopting a cylindrical shape. Also, a larger percentage of apolar residues can be accommodated in the centre of a globular unit than in a cylindrical unit. This review is primarily concerned with attempting to understand how the long, rod-shaped molecules found in fibrous proteins pack together in three dimensions in a regular or quasi-regular manner to produce functionally useful filaments or fibrils such as are present in muscle, tendon, cartilage, skin etc. Since it is often possible to produce synthetic filaments by self-assembly of the long molecules from solution, the structure of the molecular aggregate must be specifically related to the packing of the individual molecules. Electron microscopy has allowed the visualization of orderly arrays of filaments in muscle and hair but technical limitations still prevent easy recognition of regular packing of the molecules themselves. X-ray diffraction, however, conclusively shows the existence of such regular assemblies of molecules in the native state of many biological fibres. Long rodshaped macromolecules are specifically designed by nature to aggregate. The kind of molecular interactions in the aggregate are normally dictated by thermodynamic considerations of free energy. In the event of the possible formation of several structures of similar free energy, that which is biologically useful must be preferred. The purpose of a structural investigation of a fibrous protein is to see how the molecular structure can lead to specific interactions which produce regular aggregates and also how these interactions bestow properties appropriate to the biological role on the resulting aggregate. In this review we draw attention to a particular aspect of regular arrays of fibrous proteins, their synthetic analogues and also the nucleic acids, viz. statisticality. We first describe the various possible categories of statisticality and show how the X-ray diffraction patterns from such structures may be calculated. Then follows a review of the known occurrences of statisticality in assemblies of biopolymers and their analogues and a discussion of the biological implications. We also point out the characteristic features of X-ray diffraction patterns from structures which exhibit statistical ordering.

Structural studies on the tropomyosin/troponin complex of vertebrate skeletal muscle

Summary It has been shown that there is a 29A periodicity in the C-terminal half of the sequence of rabbit tropomyosin. This period, which is found in the surface apolar residues and in some of the positively and negatively charged residues, may be related to the axial separation of actin molecules in the thin filaments of vertebrate skeletal muscle. The types of interactions between tropomyosin and actin during the contraction/relaxation process are suggested. It has also been shown that the two chains of tropomyosin are most probably staggered with respect to one another by either 7 or 14 residues. Troponin appears to bind to tropomyosin either 100 or 150A from the N terminus of tropomyosin indicating that the homologous cysteine sequence is not implicated directly in the tropomyosin/troponin interaction.

Influence of dehydration method on the adsorption of benzene vapor by dried casein

Washed casein micelles were dehydrated by serial transfer through solvents of decreasing polarity followed by drying in vacuo. The product adsorbed approximately three times as much benzene as lyophilized casein for all P P 0 values at 24 and 30°C. Adsorbed C 5H 6 molecules are very mobile on the surfaces of the lyophilized particles, − q st = 8.7 kcal/mole over the entire isotherm. However, much of the benzene is in a more tightly bound adsorbed state on solvent transfer dehydrated casein, with − q st gradually decreasing from a high of 20 kcal/mole to a constant value near 8 kcal/mole. Both types of casein adsorbed more C 6H 6 than anticipated from BET surface areas calculated from low temperature N 2 adsorption data. The results suggest that the apolar amino acid residues of the casein subunits, which are normally squeezed into cavities in water according to theories of hydrophobic bonding, may be separated when water is replaced with a less polar solvent. Ultimately a porous dried product is thus obtained with localized binding sites for benzene, rather than hydrophobic regions in the macromolecule.

The covalent structure of collagen. Amino acid sequence of the N-terminal region of α2-CB4 from calf and rat skin collagen

Elucidation of the primary structure of collagen has rapidly progressed in recent years (reviewed by Traub and Piez [l] . Thus, approx. 65% of the ammo acid sequence of the cul-chain have been established [l-9]. The distribution of methionyl residues within the a2-chain is much less favourable, cyanogen bromide cleavage giving rise to three very large and three very small peptides. The large peptides are of equal length of approx. 330 amino acid residues, and the three short peptides comprise three, fourteen and thirty residues, respectively [lo]. So far, only the sequences of the small peptides have been determined [ 1, 11,121. The automated degradation procedure introduced by Edman and Begg [ 131 now rendered the large peptides likewise amenable to sequence work. In the present communication we report the sequence of the first 42 N-terminal ammo acid residues of (u2-CB4 from calf and rat skin collagen [ 10, 141. The entire peptide (112~CB4 comprises 330 residues and extends over the N-terminal third of the cr2-chain [ 14, 151. Comparison of these sequences of rat and calf collagen revealed four interspecies substitutions, all of them involving apolar residues. We also compared the first 60 N-terminal residues of the aland a2-chains of rat skin collagen.

Specific conformations and interactions in chicken erythrocyte histone F2C.

THE close association of histones with DNA in the eukaryote chromosome has implicated them in two possible functions; first, a structural role in maintaining and controlling the conformations of the chromosome through the cell cycle and second, involvement in genetic control mechanisms1. The relatively small number of histones and the rigid conservation of sequence found for histone F2A1 (see ref. 2) and suspected for other histones argue more for a structural role and involvement in the gross repression of inactive genes than for a role in the precise control of active genes. The sequence studies of histones2–6 have delineated well defined regions of the chain, rich either in basic and helix-destabilizing residues or rich in apolar, acidic and aromatic residues. Physical studies7–11 have demonstrated that the later regions are the sites of secondary structure and histone-histone interaction, while the former are the sites of DNA interaction. Thus, although particular regions of the histone chains have been correlated with quite different functions, there has been no evidence so far to show that the relatively non-basic segments fold up so specifically as in globular proteins. If, however, histones are involved in controlling the conformation of chromosomes, then specific and reversible interactions are to be expected. The very lysine-rich histones are distinguished from other histones in that microheterogeneity has been found for F1 (refs. 12–14), while F2C is polymorphic15 and it has been suggested that they have a function additional to structural. Histone F2C is unique to nucleated erythrocytes16, largely replacing F1 therein and could be involved in the total repression of erythrocyte genes.

Proton magnetic resonance studies of conformational changes n cleaved halves of histone F2B.

THE amino-acid sequences of the histones F2A11 and F2B2 are of particular interest in that they have such a high degree of non-uniformity that different regions of the polypeptide chains have quite different characters. The amino-halves of the molecules have a high density of basic residues while the carboxyl-halves have far fewer basic residues and higher proportions of apolar residues and other residues which favour the formation of helical conformations. These properties have led to the suggestions that the regions of high basicity are the primary sites for interaction with DNA1–4 in chromatin and that the non-basic regions contain any secondary structure that the molecule is capable of forming1–4 and are the sites for histone–histone interactions3,4. Evidence in support of this scheme has been obtained from nuclear magnetic resonance and optical spectroscopic studies of conformational changes and interactions in histones F1 and F2A13 and F2B4. In these studies, however, the whole molecule has been examined and the possibility of the formation of some secondary structure in the basic region of the molecule cannot be excluded.