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The synthesis of a stable substrate for enzymatic Nα-acetylation is described. To this end an analogue of α-MSH-(1–10)-decapeptide with norleucine instead of methionine at position 4 is prepared. t-Butylation of an intermediate Z-Tyr-OMe leads only to about 75% conversion in a few hours' reaction time, and cannot be carried to completion. The [Nle4]-decapeptide is as good a substrate for enzymatic Nα-acetylation as the original decapeptide containing methionine.

The 3-dimensional organisation of crystallin polypeptides into globular proteins and their interactions into higher order structures are important factors governing optical functions related to refraction, accommodation and transparency. Single crystal X-ray diffraction studies have revealed the tertiary and quaternary structural organisation of β-, γ- and δ-crystallins. Regions of the lens with high refractive index contain high levels of monomeric y-crystallins while the accommodating, hydrated avian lens has largely replaced γ-crystallins with δ-crystallin. The βγ-crystallins form a superfamily of proteins of high symmetry and great diversity in which the basic building block is a 10 kD pseudo-symmetrical 2-Greek key domain. A γ-crystallin comprises two of these β-sheet domains, joined by a linker, and a short C-terminal extension. In β-crystallins the linker has an extended conformation resulting in dimer formation by a mechanism known as domain swapping. Crystallographic analysis of engineered single domains of γ-crystallins, analogous to the ancestral domain, has indicated the importance of the short C-terminal extension in directing domain pairing. γ-crystallins have numerous cysteine residues, some are conserved in the core of the protein molecule and some are variable on the protein surface. The structure of γB-crystallin has been determined at very high resolution using cryo-crystallography allowing the visualisation of the complete protein-protein and protein-water structure at the surface. β-crystallins are seen as tetramers in the crystal structures but their long sequence extensions are harder to visualise in the electron density of the hydrated crystal lattice structure. In one tight packing lattice of βB2 crystallin the N-terminal extension is seen to mediate protein-protein interactions between tetramers to form a 42 helix. The X-ray structure of the taxon-restricted avian δ-crystallin shows that the 50 kD subunit contains 22 helices that form three α-helical domains which dimerise followed by a dimer-dimer interaction to form a tetramer with a 20-helix bundle at the centre. Analysis of the spatial disposition of the sequence conserved regions showed the location of the active site cleft of the superfamily of enzymes related to δ-crystallin and argininosuccinate lyase. A different crystal structure of δ-crystallin solved under more physiological conditions revealed that tetramers assembled as higher order supramolecular helices and that the N-terminal extension may be involved. Combining the observations of higher order helical structures in both the oligomeric β-crystallin and δ-crystallin crystal lattices, we have proposed a highly speculative model for crystallin assembly in the lens fibre cells.

The crystal structure of bovine lens gammaIIIb-crystallin at 2.5 A resolution previously reported was interpreted using a consensus sequence derived from related vertebrate sequences on the assumption that gammaIIIb-crystallin derived from the gammaC-crystallin gene. It has recently been shown that gammaIIIb is a product of the bovine gammaD gene. The structure of gammaIIIb has now been refined with the bovine gammaD sequence using new 1.95 A resolution synchrotron data. The crystallographic R factor was 20.4% for all 33 104 reflection data between 8.0 and 1.95 A measured at 277(1) K. The electron density fully supported the assignment of the gammaD sequence to gammaIIIb. The crystal belongs to space group P2(1)2(1)2(1) with two molecules of molecular mass 20 749 Da in the asymmetric unit in which 219 water molecules were located. The two-domain four-Greek-key motif highly symmetrical protein is very similar in structure to gammaB-crystallin (81% sequence identity). There is a single amino-acid deletion in gammaD in the linker region connecting the two domains. The intermolecular oganization in the crystal lattice is quite different from gammaB as a result of key mutations involving surface residues Leu51, Ile103 and His155. These point mutations will contribute to the intermolecular behaviour of the gamma-crystallins in the eye lens, where they are major components of the densely packed, high refractive index regions of the lens.

We use protein engineering and crystallography to simulate aspects of the early evolution of beta gamma-crystallins by observing how a single domain oligomerizes in response to changes in a sequence extension. The crystal structure of the C-terminal domain of gamma beta-crystallin with its four-residue C-terminal extension shows that the domain does not form a symmetric homodimer analogous to the two-domain pairing in beta gamma-crystallins. Instead the C-terminal extension now forms heterologous interactions with other domains leading to the solvent exposure of the natural hydrophobic interface with a consequent loss in protein solubility. However, this domain truncated by just the C-terminal tyrosine forms a symmetric homodimer of domains in the crystal lattice.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins. The main sequence difference between the two families is the presence of sequence extensions in the beta-crystallins. A major question concerns the role that these extensions play in mediating interactions at the high protein concentrations found in the lens. The predominant beta-crystallin polypeptide, beta B2, can be crystallized in two different space groups, I222 and C222. The I222 crystal structure revealed that the protein packed as a tetramer with perfect 222 symmetry but that the extensions were disordered. The X-ray structure of the C222 lattice of beta B2 has now been refined at 3.3 A, the structure analysed and compared with the I222 lattice. The protein is also a tetramer with 222 symmetry in the C222 lattice but differs in that parts of the N-terminal extensions have been visualized. In the asymmetric unit of the C222 lattice there are four subunits, each comprising a single polypeptide chain, in which certain flexible loops in the N-terminal domains and the N-terminal extensions have various conformations. The tetramers in the C222 lattice are more tightly packed than in the I222 form. Analysis of the tetramer contacts shows that the sites of interaction break the 222 symmetry of the tetramers. The N-terminal extensions play a major role in directing interactions between tetramers. One of the N-terminal extensions interacts with a hydrophobic patch on the N-terminal domain of another tetramer. These crystallographic observations obtained over a physiological concentration range indicate how, in beta-crystallin oligomers, the N-terminal extensions of beta B2 can switch from interacting with water to interacting with protein depending on their relative concentrations. This could be useful in maintaining a gradient of refractive index.

The molecular structure of calf γB-crystallin (previously called γII), a lens-specific protein, has been refined to a crystallographic R factor of 18.1% for all reflection data, between 8.0 and 1.47 A, 25 959 hkl measured at 293 (1) K. 230 water molecules have been defined by difference Fourier techniques and included in a restrained least-squares refinement. Difference Fourier maps clearly indicated the presence of multiple sites for the sulfur atoms of Cys 18 and Cys 22 which were therefore given coupled second-site occupancies during the refinement. The sulfur atom in the major position of Cys 22 is in the reduced state. Either of the Cys 18 sites can form a high-energy disulfide bridge with the minor position of Cys 22. The position of the carboxy terminus and many other surface side chains have been further defined including the RGD signal peptide. The hydration of the backbone and the interdomain region has been analysed. 27 water molecules make extensive contacts to a single protein molecule and thus contribute to its stability.

Annexin VI is an eight repeat member of the annexin family of proteins which are both water soluble and bind to negatively charged phospholipids in a calcium-dependent manner. Here we present a model for annexin VI based on fitting the three-dimensional structure of two annexin V molecules (Huber (1990) EMBO J. 9, 3867–23;874) to the two-dimensional stain-excluding density of lipid-bound annexin VI (Newman (1989) J. Mol. Biol. 206, 213–219). Both annexin VI lobes could only be fitted with their convex faces closest to the lipid monolayer. This supports the hypothesis that annexin—lipid binding is mediated by the interaction between calcium bound to the loops protruding from the convex protein surface and phospholipid headgroups.

X-ray analyses have defined the three-dimensional structures of crystals of mouse and human renins complexed with peptide inhibitors at resolutions of 1.9 and 2.8 A, respectively. The exquisite specificity of renin arises partly from ordered loop regions at the periphery of the binding cleft. Although the pattern of main-chain hydrogen bonding in other aspartic proteinase inhibitor complexes is conserved in renins, differences in the positions of secondary structure elements (particularly helices) also lead to improved specificity in renins for angiotensinogen substrates.

β-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of βB2 dimer has been solved at 2·1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar “Greek key” motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their β-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of γ-crystallins are monomers, β-crystallins are always oligomers. In the βB2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of βB2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of γB-crystallin. The intersubunit domain interface is very similar to the intradomain interface of γB, although many sequence differences have resulted in an increase in polar interactions between domains in βB2. Comparison of the structures of βB2 and γB-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the β-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens

Except for the two acidic chains, βA2 and βA4, the primary structures of all bovine β-crystallins have previously been elucidated, either by direct protein sequencing or prediction from cDNA sequencing. Both βA2 and βA4 were found to be synthesized in half-year-old calf lenses and are therefore likely to be present in a cDNA bovine library constructed from mRNA isolated from lenses of that age. A large number of cDNA clones was screened with all available crystallin, actin, vimentin and lens membrane protein MP26 probes and finally with a randomly primed mRNA probe. Clones positive for the latter, but negative for known lens proteins, were isolated and sequenced. βA2, comprising 197 aa, and βA4, comprising 209 aa, were identified. Both proteins have a conserved two-domain structure and an N-terminal extension which is variable. A three-dimensional model of the structure of βA4 was made based on the coordinates of one subunit from the βB2 dimer which has recently been solved using x-ray diffraction techniques. The resulting heterodimer structure, together with the compiled bovine β-crystallin sequences, was used to indicate those regions of the sequences which distinguish acidic from basic β-crystallins with a view to defining structural features necessary for subunit recognition in β-crystallin aggregates. With the aid of the present data, the complete evolutionary tree of the bovine β-crystallin family has been constructed, which confirms the early separation of the genes encoding the three acidic and the three basic β-crystallins.

The iron-dependent superoxide dismutase from Mycobacterium tuberculosis has been crystallized by the hanging drop method. The crystals belong to the P2(1) space group and have unit cell dimensions of a = 68.5 A, b = 85.6 A, c = 66.5 A, beta = 99.8 degrees. There are four molecules per asymmetric unit which, from analysis of data to 2.5 A, appear to be related by non-crystallographic 222 symmetry.

The transparency of the lens depends on an even distribution of protein and water over distances comparable to the wavelength of light, while the degree of refraction is controlled partly by the ability of the lens to change shape. The core regions of certain lenses such as carp and rat have an extremely high refractive index as a result of high protein concentration, which confers rigidity on that region of the lens. By contrast the outer regions of these lenses, like the complete human lens, have a lower proportion of protein to water and are malleable (van Heyningen, 1976; Philipson, 1969; Fagerholm et al., 1981). Furthermore, there is an increasing protein concentration gradient from the periphery to the core of the lens, leading to a gradient of refractive index that almost abolishes spherical aberration (Fernald and Wright, 1983; Sivak, 1985).

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.

γ-Crystallins are a family of low molecular weight proteins found in high concentration in the densely packed regions of high refractive index in vertebrate lenses. Certain members have the characteristic property of a high critical temperature (tc) for phase separation. We report the three-dimensional structure determination of such a protein, bovine lens γIVa-crystallin, which has been refined to give an X-ray R-factor of 0.143. Its high tc contrasts with the low tc γII-crystallin, whose structure we have already published. The root mean square difference between the α-carbon atoms of these two proteins is 0.70 A and γIVa has an internal symmetry even higher than that of γII. The presence of a protein that exhibits the phenomenon of phase separation at body temperature renders the lens very susceptible to a transformation from transparent to an opaque state due to irregularities in the refractive index. Protein interactions of γIVa-crystallin have implications for the mechanism of cataract formation. Modes of self-association behaviour of γIVa-crystallin have been inferred from an analysis of the lattice interactions in the crystalline state, where the protein packing density is similar to that of the intact lens. It appears that the point mutation at position 103 from a serine residue in γII to a valine in γIVa gives rise to a lattice contact formed by two four-stranded β-sheets in γIVa. A group-specific mutation at position 118 from leucine to phenylalanine induces subtle differences in core packing, leading to a reorganization around residue 103. However, the final phase separation determinant may be a complex combination of many side-chain functions.

N alpha-acetylation is almost exclusively restricted to eukaryotic structural proteins. As a rule it is a post-initiational process, requiring the presence of the enzyme N alpha-acetyltransferase and the acetyl donor acetylcoenzyme A. N alpha-acetyltransferases appear to have a narrow substrate specificity, which is very similar for enzymes from different tissues and species. Amino acids predominantly present at the N terminus of N alpha-acetylated proteins are alanine, serine, and methionine. The occurrence of these residues is apparently a prerequisite for acetylation. The region following these amino acids is also important. If methionine is at the N terminus, the second position is always occupied by a strongly hydrophilic amino acid. Two- and three-dimensional structural characteristics of the protein do not seem to play a major role in N alpha-acetylation. Up to now the exact function for N alpha-acetylation is not known.

Four recombinant cDNA clones coding for a 23 kDa beta-crystallin polypeptide of the frog (Rana temporaria) were identified in a collection of cloned cDNA and two of them were sequenced. The cDNA present in these clones codes for a polypeptide 198 amino-acid residues in length, which appears to be the frog beta A1-crystallin because of its high homology with the sequences of beta A1-crystallins from other species. Furthermore, the nucleotide sequence coding for the compact folded region of the protein is highly conserved. Virtually no homology was found in the 3' nontranslated regions of the mRNA. The amino-acid sequence of the Rana beta A1-crystallin was used to build a three-dimensional model based on the coordinates of the homologous bovine gamma II. An analysis of the model shows that the surface residues of the beta A1-crystallin (amphibian, mammalian and bird) are more highly conserved than the buried residues. It is suggested that this is related to the oligomeric nature of the lens beta-crystallins.

A rapid and specific assay for Nα-acetyltransferase activity is described. This assay makes use of acidic precipitation for removal of products resulting from acetylation of endogenous substrates from the enzyme preparation and of reverse-phase HPLC for isolation of the N-terminally acetylated synthetic substrate. The virtue of the assay lies in its fast, not too laborious, and reasonably accurate determination of activity during isolation and purification of the enzyme.

beta-Crystallins are complex oligomers composed of many related subunits. In order to understand their interactions we have built molecular models of several bovine beta-crystallins, based on their sequence similarity to the well-defined gamma-II crystallin structure, using interactive computer graphics techniques. Their common origin with gamma-crystallin is displayed in both the retention of four-fold sequence repeats of critical residues involved with stabilizing a folded beta-hairpin and the conservation of core-filling hydrophobic side-chains. The beta-crystallins have been built as bilobal molecules with each domain composed of two 'Greek key' motifs which associate about an approximate two-fold axis to form beta-sheets. The beta-crystallin sequences have previously been shown to comprise two families, the basic and acidic subunits, which have extensions of sequence. The three-dimensional models show how the two families appear to stabilize the folded beta-hairpin in the N- and C-terminal domains in ways which suggest that they have diverged from a common ancestor in different ways. Acidic beta-crystallins, like gamma-crystallins, have a regular array of charges on their N-terminal domain which has been interrupted in basic beta-crystallins by hydrophobic residues which may be related to the presence of a C-terminal extension. beta-Crystallins are more highly charged than gamma-crystallins although their charge density is higher in certain regions of the N-terminal domain, particularly in beta B1-crystallin. beta-crystallins also differ from gamma-crystallins in the virtual absence of core-filling sulphydryl groups whereas they have numerous sulphur-containing side-chains together with tryptophan and histidine rings protruding from the globular domains, particularly in the acidic subunits. The burial of these residues in subunit contacts is consistent with their spectroscopic and electrostatic properties. Protein subunit aggregation commonly occurs through hydrophobic interaction or beta-sheet extension. Analysis of the subunit surfaces has identified an N-terminal hydrophobic region common to beta B1 and beta B2 whereas a C-terminal hydrophobic loop region is common to beta B1 and beta A1 and may be correlated with their association properties. It is suggested that the polar C-terminal domain of beta B2 contributes towards the solubility of higher aggregates by interactions involving beta-sheet structure.