Main-chain Fold Research Articles

Structure of Hen Lysozyme in Solution

The structure of the 129-residue protein hen lysozyme has been determined in solution by two-dimensional 1H nuclear magnetic resonance methods. 1158 NOE distance restraints, 68 φ and 24 χ 1 dihedral angle restraints were employed in conjunction with distance geometry and simulated annealing procedures. The overall C α root-mean-square deviation from the average for 16 calculated structures is 1·8(±0·2)Å, but excluding 14 residues in exposed disordered regions, this value reduces to 1·3(±0·2) Å. Regions of secondary structure and the four α-helices in particular, are well defined ( C α root-mean-square deviation 0·8(±0·3) Å for helices). The main-chain fold is closely similar to structures of the protein in the crystalline state. Furthermore, many of the internal side-chains are found in well-defined conformational states in the solution structures, and these correspond well with the conformational states found in the crystal. The general high level of definition of main-chain and many internal side-chains in the solution structures is reinforced by the results of an analysis of coupling constants and ring current shifts. Many side-chains on the surface, however, are highly disordered amongst the set of solution structures. In certain cases this disorder has been shown to be dynamic in origin by the examination of 3 J αβ coupling constants.

Structural repertoire of the human V H segments

The V H gene segments produce the part of the V H domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human V H segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 V H segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the V H segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the V H segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.

Crystal structure of staphylococcal enterotoxin B, a superantigen.

The three-dimensional structure of staphylococcal enterotoxin B, which is both a toxin and a super-antigen, has been determined to a resolution of 2.5 A. The unusual main-chain fold containing two domains may represent a general motif adopted by all staphylococcal enterotoxins. The T-cell receptor binding site encompasses a shallow cavity formed by both domains. The MHCII molecule binds to an adjacent site. Another cavity with possible biological activity was also identified.

Expression, purification, and characterization of a recombinant ribonuclease H from Thermus thermophilus HB8.

Thermus thermophilus ribonuclease H was overexpressed and purified from Escherichia coli. The determination of the complete amino acid sequence allowed modification of that predicted from the DNA sequence, and the enzyme was shown to be composed of 166 amino acid residues with a molecular weight of 18,279. The isoelectric point of the enzyme was 10.5, and the specific absorption coefficient A0.1%(280) was 1.69. The enzymatic and physicochemical properties as well as the thermal and conformational stabilities of the enzyme were compared with those of E. coli RNase HI, which shows 52% amino acid sequence identity. Comparison of the far and near UV circular dichroism spectra suggests that the two enzymes are similar in the main chain folding but different in the spatial environments of tyrosine and tryptophan residues. The enzymatic activities of T. thermophilus RNase H at 37 and 70 degrees C for the hydrolysis of either an M13 DNA/RNA hybrid or a nonanucleotide duplex were approximately 5-fold lower and 3-fold higher, respectively, as compared with E. coli RNase HI at 37 degrees C. The melting temperature, Tm, of T. thermophilus RNase H was 82.1 degrees C in the presence of 1.2 M guanidine hydrochloride, which was 33.9 degrees C higher than that observed for E. coli RNase HI. The free energy changes of unfolding in the absence of denaturant, delta G[H2O], of T. thermophilus RNase H increased by 11.79 kcal/mol at 25 degrees C and 14.07 kcal/mol at 50 degrees C, as compared with E. coli RNase HI.

Crystal structure of α-dendrotoxin from the green mamba venom and its comparison with the structure of bovine pancreatic trypsin inhibitor

The three-dimensional structure of α-dendrotoxin (α-DTX) from the green mamba ( Dendroaspis angusticeps) venom has been determined crystallographically using the method of isomorphous replacement and refined at 2.2 Å resolution using a restrained least-squares method. The crystallographic R-factor is 0.169 for all 3451 measured reflections between 7.0 and 2.2 Å. Although the main-chain fold of α-DTX is similar to that of homologous bovine pancreatic trypsin inhibitor (BPTI), there are significant differences involving segments of the polypeptide chain close to the “antiprotease site” of BPTI. Comparison of the structure of α-DTX with the existing models of BPTI and its complexes with trypsin and kallikrein reveals structural differences that explain the inability of α-DTX to inhibit trypsin and chymotrypsin.

Crystallization and Preliminary X-Ray Structure Analysis of Pigeon Egg-White Lysozyme1

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.

Amino acid similarity coefficients for protein modeling and sequence alignment derived from main-chain folding angles

A set of “similarity-parameters” was calculated that reflects the influence of the proteinogenic amino acids on the structure of the protein backbone. The parameters were derived from a detailed analysis of the amino acid specific main-chain torsion angle distributions as they are found in proteins (highly resolved protein structures from the Brookhaven Protein Data Bank). The purpose of these parameters is threefold: (1) they should help in estimating the structural effect of an amino acid substitution during the design of new mutants in protein-engineering; (2) in modeling by homology they should mark places in the protein where changes in the folding are expected; and (3) they should form a scoring matrix in protein sequence alignment superior to identity scoring. The usability of the “structure derived correlation matrix (SCM)” for these purposes is assessed and demonstrated for some examples in the paper.

Analysis of sequence-similar pentapeptides in unrelated protein tertiary structures: Strategies for protein folding and a guide for site-directed mutagenesis

From the most recent Brookhaven Protein Co-ordinate Databank, 229 sequence-identical pentapeptide pairs, each found in two unrelated protein structures, were collected; 9115 such pairs differing in only one residue were also gathered. For both samples the main-chain fold was conserved about 20% of the time, despite the different atomic environments presented by the unrelated protein architectures. An analysis of the substituted residues as well as the composition of the sequence-similar pentapeptides allowed several suggestions regarding protein folding mechanisms. An examination of the most frequently observed residue substitutions and their correlation with structural changes in the oligopeptide pairs yields a possible guide for site-directed mutagenesis experiments, especially when no tertiary structural information is at hand.

Main chain fold of a [2Fe-2S]ferredoxin I from Aphanothece sacrum at 2.5 A resolution.

Crystal structure analysis of a [2Fe-2S]ferredoxin I from Aphanothece sacrum, a blue green alga, was carried out at 2.5 A resolution. The phase angle of each reflection was determined by the single isomorphous replacement method coupled with the anomalous dispersion effect for the uranium derivative. The four molecules in an asymmetric unit were clearly seen in a 3.9 A electron density map. The main chains of three molecules were traced at 2.5 A resolution. The structure of the remaining one molecule, however, remains unknown because of the poor electron density of the corresponding region. The three main chain folds exhibit the same topology as that in Spirulina platensis. The structural similarity between A. sacrum and S. platensis ferredoxins, whose amino acid sequences are different from each other by about 30%, strongly suggests that all plant-type ferredoxins have the same main chain fold.

Comparison of the folding of 2-Keto-3-deoxy-6-phosphogluconate aldolase, triosephosphate isomerase and pyruvate kinase: Implications in molecular evolution

The 8-fold α/β barrel conformation of 2-keto-3-deoxy-6-phosphogluconate aldolase has been compared to that of triosephosphate isomerase and the A-domain of pyruvate kinase. There are eight supersecondary structure units (α/β) in each of these proteins, and the comparisons were carried out in orientations corresponding to each of the possible congruences, i.e. first to first, first to second,… of the supersecondary structure units. The comparison of the C α structure of the main chain folding of the three enzymes indicated about 150 equivalences with rootmean-square differences of about 3.1 Å, with no orientational preference, including the aldolase with itself. In addition, there is no sequence homology between the aldolase and the isomerase, and no indication of gene duplication in the former. The lack of orientational preference among the three enzymes suggests convergence to a fold of exceptional stability. However, all three enzymes activate a CH bond adjacent to a carbonyl, and their active sites correspond to the f strand, F helix region of the α/β barrel, thus contradicting the foregoing and suggesting divergent evolution from a common precursor. Other and similar arguments are also presented for and against convergent evolution of these three strikingly similar enzymes.

X-ray analysis of a [2Fe-2S] ferrodoxin from Spirulina platensis. Main chain fold and location of side chains at 2.5 A resolution.

A [2Fe-2S] ferrodoxin from Spirulina platensis crystallized in space group C2221 with cell dimensions of a = 62.32, b = 28.51, c = 108.08 A, and alpha = beta = gamma = 90.0 degrees. X-ray structure analysis of the protein was carried out at 2.5 A resolution by the single isomorphous replacement method coupled with the derivative and the native anomalous dispersion methods. Phase angles of 2182 independent reflections were determined and their average figure of merit was 0.58. Each of 98 residues was superposed on the electron density sections enlarged to 2 cm/l A with a half-mirror device (Richards box). About 25% of the total residues form beta-structure and 10% fold in a tow-turn alpha-helix. A beta-barrel-like structure was found in the main chain fold. A polypeptide segment from residues 41 to 49 forms a loop structure outside the barrel. Two iron atoms of the [2Fe-2S] cluster are coordinated by three cysteines in the loop and by Cys-79. Hydrogen bonds of NH....S and OH....S stabilize the loop conformation. Most side chains are reasonably oriented in the molecule. The internal volume of the barrel is occupied by aliphatic nonpolar residues. All the charged groups are accessible to solvent molecules.

On cytochrome c3 folding.

The structure of cytochrome c3 from Desulfovibrio vulgaris Miyazaki (DvM) is considered in detail by scrutinizing main chain folding together with the structure of the protein from D. desulfuricans Norway (DdN). The relative arrangement of the four heme groups in this molecule is similar to that of DdN and the disposition of alpha-carbon atoms of cysteine and histidine residues binding to heme groups is also similar. Structural differences between the two proteins occur in the shape of some specific loops of the chain on the molecular surface. As a result of a careful comparison of structures and sequences in both cytochromes c3, the reported sequence alignment of the cytochrome c3 family has been revised. Our new proposal of a sequence alignment based on the three-dimensional structures contains 24 evolutionarily conservative residues. All these conservative residues may play an important role in the folding pattern of cytochromes c3.

Micellar solubilization of biopolymers in hydrocarbon solvents. ii. the case of horse liver alcohol dehydrogenase

The chemical characterization of horse liver alcohol dehydrogenase solubilized in isooctane via reverse micelles formed by the anionic surfactant di (2-ethyl-hexyl) sodium sulfosuccinate (AOT) and water (0.6 to 4% v/v) is presented. The enzyme’s catalytic activity toward acetaldehyde reduction is markedly dependent upon w0 = [H2O]/[AOT], and upon the pH of the stock aqueous solution (pHst), from which the hydrocarbon enzyme solution is prepared. Kinetically, the micellar solution appears to follow a normal Michaelis-Menten behavior, with a turnover number which, under the optimal conditions (w0 = 42, pHst = 8.8), appears to be higher than in bulk water. The affinity between enzyme and NADH, as judged from direct binding studies (quenching of the protein fluorescence), is much reduced with respect to water if concentrations refer to the water pool of the micelles, and comparable to water if concentrations refer to the overall volume (hydrocarbon plus water pool). Also, the Km values are much higher if concentrations refer to the water pool. Ultraviolet absorption studies show that the aromatic chromophores are not significantly perturbed on going from a water solution to the micellar solution. The essentially aqueous environment of the protein in the reverse micelles is confirmed by fluoresence studies. Circular dichroism studies show that the enzyme’s conformation in the micelles is similar to that in water; however, under certain conditions, small but significant changes of the main chain folding seem to occur, which do not impair enzymatic activity. The spectroscopic properties of NADH in the hydrocarbon phase (fluorescence and circular dichroism) are also investigated. The potential of the LADH-NADH system for technical applications (oxidoreduction of lipophylic substrates) is discussed.

Prediction of the secondary and tertiary structure of plastocyanin.

The amino acid sequences of nine plastocyanins were examined using four published methods for the prediction of secondary structure in proteins. The results of the four methods were combined in such a way as to maximize agreement, and the position of alpha helices, beta sheets, and beta turns in plastocyanin was predicted. From this result and other information, such as the position of conserved residues and the requirements for coordination of copper, a preliminary model for the mainchain folding of the molecule was presented.