Conformational analysis of thymidylate synthase from amino acid sequence and circular dichroism

Circular dichroism studies were carried out in the vacuum ultraviolet region for thymidylate synthase from Lactobacillus casei and its ligand complexes. The CD spectrum was analyzed for secondary structure by our method and the variable selection method, and both gave similar results. Our method predicts 33% alpha-helix, 25% (23% antiparallel and 2% parallel) beta-sheet, 20% turns, and 16% other structure. The secondary structure of this protein was also predicted from the amino acid sequence by four different methods. Though there is a variation in the prediction among these methods, the prediction of 32% alpha-helix and 23% beta-sheet by combining the four methods is in excellent agreement with our CD results. Further, the location of the predicted regions of alpha-helices and beta-strands along the sequence and the CD characteristics strongly suggest that this protein belongs to an alpha + beta structural class. Binding of the inhibitor FdUMP or the cofactor 5,10-methylenetetrahydrofolate did not change the CD spectrum. However, when both ligands were present, there was a significant change in the CD spectrum and the maximum changes occurred when the concentration of FdUMP was 1 mol/mol of enzyme. The addition of FdUMP and cofactor causes, respectively, a 5% and 6% decrease in beta-sheet and beta-turns and about an 8% increase in "other" structure.     

The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.     

Circular dichroism studies of acetylcholinesterase conformation. Comparison of the 11 S and 5.6 S species and the differences induced by inhibitory ligands

Circular dichroism studies were carried out in the vacuum ultraviolet region for 11 S and 5.6 S species of acetylcholinesterase from Torpedo. As the 5.6 S acetylcholinesterase forms larger oligomers in the absence of detergent, the CD spectrum was measured both with and without detergent. Secondary structure analysis of the CD spectrum for 11 S acetylcholinesterase shows 33% alpha-helix, 23% beta-sheet (14% antiparallel and 9% parallel), 17% turns and 26% other structure. Binding of edrophonium to the active site of 11 S acetylcholinesterase increases alpha-helix, while binding of propidium to the peripheral site increases beta-sheet. The beta-sheet content is slightly higher for 5.6 S than 11 S acetylcholinesterase in water. When the detergent is added to 5.6 S acetylcholinesterase, the 190 nm and 220 nm bands become less intense, although the analyses of the two spectra are similar. No significant change is observed for the 5.6 S form in either solvent on binding ligands. The prediction of both parallel and antiparallel beta-sheet suggests that at least one domain in these multidomain proteins belongs to the alpha/beta tertiary structural type.     

Secondary structure of the murine histocompatibility alloantigen H-2Kb: relationship between heavy chain, beta 2-microglobulin, and antigenic reactivity

The far-ultraviolet circular dichroism (CD) spectra of the extracellular portion (papain-cleaved fragment) of the histocompatibility antigen H-2Kb and its noncovalently associated components, heavy chain and beta 2-microglobulin (beta 2m), indicate that the antigen is highly structured, containing about 30% alpha-helix, 41% beta-sheet, and 29% random coil. Separation of beta 2m from the heavy chain produced a decrease in heavy chain alpha-helix and beta-sheet structure which correlated with a loss of alloantigenic reactivity. Reconstitution of the heavy chain-beta 2m complex resulted in an increase in secondary structure which was greater than the sum of the free chains and the recovery of considerable alloantigenic reactivity. This suggests that some of the secondary structure and much of the alloantigenic reactivity may depend on conformation associated with the binding of beta 2m to heavy chain. A prediction of heavy chain secondary structure based on Chou-Fasman analysis of the primary amino acid sequence agreed with results from CD measurements and suggested that the segments of alpha-helix and beta-sheet structure are distributed throughout the molecule.     

Standard conformations of a polypeptide chain in irregular protein regions

A detailed stereochemical analysis of known protein structures has been made which shows that: (1) irregular regions of proteins consist of a limited number of standard structures formed by three, four of more residues; (2) an amino acid residue of a protein can adopt one of the six sterically allowed conformations designated here as alpha, alpha L, beta, gamma, delta, and epsilon. It is shown that there are two allowed conformations of a polypeptide chain at the N-end of an alpha-helix, beta alpha n- and beta gamma alpha n-conformations, where n is a number of residues in the alpha-helix. At the C-end of the alpha-helix there are two conformations as well, alpha n gamma beta- and alpha n gamma alpha L beta-ones. Two beta-strands in a beta-hairpin can be joined, for example, by standard structures with beta beta alpha L beta-, beta alpha gamma alpha L beta-, beta alpha alpha gamma alpha L beta-conformations which are referred to as turns. In the regions where a polypeptide chain passes from one layer to another there are standard structures with beta gamma beta-, beta alpha beta beta-, beta alpha gamma beta-conformations etc., referred to as cross-overs. A structure of any protein irregular region can be represented as a combination of these and other standard turns and cross-overs considered in the paper. The major part of the turns and cross-overs has residues in alpha L- or epsilon-conformations which must be glycine or other residues with small or flexible side chains. Massive hydrophobic residues must not occupy the first beta-positions of the most standard structures. The results obtained can be successfully applied for prediction of the location of the turns and cross-overs in proteins from their amino acid sequences and for interpretation of electron density maps.     

Determination of secondary structure of globular proteins using circular dichroism spectra

A ridge regression method is presented for prediction of the secondary structure of proteins by the circular dichroism spectra (CD) from 190 to 236 nm. Eight types of the secondary structure were calculated on a microcalculator. The method is based on the X-ray data of Kabsh and Sander. The teaching rule is constructed on CD spectra of 30 proteins of all structural classes of the globular proteins (alpha, alpha/beta, alpha + beta and beta-proteins). The errors of the methods are analysed by removing each protein from the reference set and analyzing its structure in terms of the remaining proteins. Correlation coefficients and root-mean square deviations between CD and X-ray data were: 0.99 and 0.03 for alpha-helix, 0.86 and 0.02 for 3(10)-helix, 0.92 and 0.06 for antiparallel beta-sheet, 0.86 and 0.03 for parallel beta-sheet, 0.94 and 0.01 for T3 beta-turn, 0.85 and 0.02 for other beta-turn, 0.84 and 0.03 for S-bends, 0.83 and 0.04 for "random" structure.     

The predicted secondary structures of class I fructose-bisphosphate aldolases.

The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turns for fructose-bisphosphate aldolases from human and rat muscle and liver, from Trypanosoma brucei and from Drosophila melanogaster. All the aldolase sequences gave essentially the same pattern of secondary-structure predictions despite having sequences up to 50% different. One exception to this pattern was an additional strongly predicted helix in the rat liver and Drosophila enzymes. Regions of relatively high sequence variation generally were predicted as reverse turns, and probably occur as surface loops. Most of the positions corresponding to exon boundaries are located between regions predicted to have secondary-structural elements consistent with a compact structure. The predominantly alternating alpha/beta structure predicted is consistent with the alpha/beta-barrel structure indicated by preliminary high-resolution X-ray diffraction studies on rabbit muscle aldolase [Sygusch, Beaudry & Allaire (1986) Biophys. J. 49, 287a].     

Prediction of secondary structure for Eco RI endonuclease

The circular dichroism of Eco RI restriction endonuclease was measured to 178 nm and analyzed for secondary structure. The results (33% alpha-helix, 25% beta-sheet, 17% turns, and 25% other structures) compare well with our joint prediction from sequence data.     

Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction

Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of the PsbQ protein of photosystem II by Fourier transform infrared and circular dichroic spectroscopy and by bioinformatic methods

The structure of PsbQ, one of the three main extrinsic proteins associated with the oxygen-evolving complex (OEC) of higher plants and green algae, is examined by Fourier transform infrared (FTIR) and circular dichroic (CD) spectroscopy and by computational structural prediction methods. This protein, together with two other lumenally bound extrinsic proteins, PsbO and PsbP, is essential for the stability and full activity of the OEC in plants. The FTIR spectra obtained in both H(2)O and D(2)O suggest a mainly alpha-helix structure on the basis of the relative areas of the constituents of the amide I and I' bands. The FTIR quantitative analyses indicate that PsbQ contains about 53% alpha-helix, 7% turns, 14% nonordered structure, and 24% beta-strand plus other beta-type extended structures. CD analyses indicate that PsbQ is a mainly alpha-helix protein (about 64%), presenting a small percentage assigned to beta-strand ( approximately 7%) and a larger amount assigned to turns and nonregular structures ( approximately 29%). Independent of the spectroscopic analyses, computational methods for protein structure prediction of PsbQ were utilized. First, a multiple alignment of 12 sequences of PsbQ was obtained after an extensive search in the public databases for protein and EST sequences. Based on this alignment, computational prediction of the secondary structure and the solvent accessibility suggest the presence of two different structural domains in PsbQ: a major C-terminal domain containing four alpha-helices and a minor N-terminal domain with a poorly defined secondary structure enriched in proline and glycine residues. The search for PsbQ analogues by fold recognition methods, not based on the secondary structure, also indicates that PsbQ is a four alpha-helix protein, most probably folding as an up-down bundle. The results obtained by both the spectroscopic and computational methods are in agreement, all indicating that PsbQ is mainly an alpha protein, and show the value of using both methodologies for protein structure investigation.     

The conformation of glucagon: predictions and consequences.

It is proposed that glucagon, a polypeptide hormone, is delicately balanced between two major conformational states. Utilizing a new predictive model [Chou, P.Y., and Fasman, G.D. (1974), Biochemistry 13, 222] which considers all the conformational states in proteins (helix, beta sheet, random coil, and beta turns), the secondary structural regions of glucagon are computed herein. The conformational sensitivity of glucagon may be due to residues 19-27 which have both alpha-helical potential (mean value of Palpha = 1.19) as well as beta-sheet potential (mean value of Pbeta = 1.25). Two conformational states are predicted for glucagon. In predicted form (a), residues 5-10 form a beta-sheet region while residues 19-27 form an alpha-helical region (31% alpha, 21% beta) agreeing well with the circular dichroism (CD) spectra of glucagon. The similarity in the CD spectra of glucagon and insulin further suggests the presence of beta structure in glucagon, since X-ray analysis of insulin showed 24% beta sheet. In predicted form (b), both regions, residues 5-10 and residues 19-27, are beta sheets sheets (0% alpha, 52% beta) in agreement with the infrared spectral evidence that glucagon gels and fibrils have a predominant beta-sheet conformation. Since three reverse beta turns are predicted at residues 2-5, 10-13, and 15-18, glucagon may possess tertiary structure in agreement with viscosity and tritium-hydrogen exchange experiments. A proposal is offered concerning an induced alpha yields beta transition at residues 22-27 in glucagon during receptor site binding. Amino acid substitutions are proposed which should disrupt the beta sheets of glucagon with concomitant loss of biological activity. The experimental findings that glucagon aggregates to form dimers, trimers, and hexamers can be explained in terms of beta-sheet interactions as outlined in the present predictive model. Thus the conflicting conclusions of previous workers, concerning the conformation of glucagon in different environments, can be rationalized by the suggested conformational transition occurring within the molecule.     

The predicted secondary structure of enolase.

The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turn for both chicken skeletal-muscle and yeast enolase sequences. The predicted secondary-structure content of the chicken enzyme is 27% alpha-helix and less than 10% beta-sheet, whereas in the yeast enolase a similar helix content but virtually no sheet are predicted. These results are in fair agreement with published experimental estimates of the amount of secondary structure in the yeast enzyme. The enzyme appears to be formed from three domains.     

Energy of stabilization of the right-handed beta alpha beta crossover in proteins

An explanation in terms of conformational energies is provided for the observed nearly exclusive preference of the beta alpha beta structure for forming a right-handed, rather than a left-handed, crossover connection. Conformational energy computations have been carried out on a model beta alpha beta structure, consisting of two six-residue Val beta-strands and of a 12-residue Ala alpha-helix, connected by two flexible four-residue Ala links to the strands. The energy of the most favorable right-handed crossover is 15.51 kcal/mol lower than that of the corresponding left-handed cross-over. The right-handed crossover is a strain-free structure. Its energy of stabilization arises largely from the interactions of the two beta-strands with one another and with the alpha-helix. On the other hand, the left-handed crossover is either disrupted after energy minimization or it remains conformationally strained, as indicated by an energetically unfavorable left twisting of the beta-sheet and by the presence of high-energy local residue conformations. In the energetically most favorable right-handed crossover, the right twisting of the beta-sheet and its manner of interacting with the alpha-helix are identical with those computed earlier for isolated beta-sheets and for packed alpha/beta structures. This result supports a proposed principle that it is possible to account for the main features of frequently occurring structural arrangements in globular proteins in terms of the properties of their component structural elements.     

Determination of protein secondary structure using factor analysis of infrared spectra

A method is presented for determining the secondary structural composition of a protein in aqueous solution from its infrared spectrum. A factor analysis approach is used to analyze the infrared spectra of 18 proteins whose crystal structures are known from X-ray studies. Factor analysis followed by multiple linear regression identifies those eigenspectra that correlate with the variation in properties described by the calibration set. The properties of interest in this study are % alpha-helix, % beta-sheet, and % turns. In the analysis of an unknown, the factor loadings required to reproduce its spectrum are substituted in the regression equation for each property to predict its secondary structural composition. The accuracy of the method was determined by removing each standard, in turn, from the calibration set and using a calibration set generated from the remainder to predict its composition. By this method we obtain standard errors of prediction of 3.9% for alpha-helix, 8.3% for beta-sheet, and 6.6% for turns. The method may also be applied to the spectra of proteins in 2H2O. The method has important advantages over those currently in use for the quantitative analysis of the infrared spectra of proteins. Manipulation of the spectrum is kept to a minimum, no curve-fitting is necessary, and the several amide I band components need not be assigned.     

Analysis of interactive packing of secondary structural elements in alpha/beta units in proteins

An alpha-helix and a beta-strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element. An analysis of all such 5,975 nonidentical alpha/beta units in protein structures, defined at < or = 2.5 A resolution, shows that the interaxial distance between the alpha-helix and the beta-strand is linearly correlated with the residue-dependent function, log[(V/nda)/n-int], where V is the volume of amino acid residues in the packing interface, nda is the normalized difference in solvent accessible contact area of the residues in packed and unpacked secondary structural elements, and n-int is the number of residues in the packing interface. The beta-sheet unit (beta u), defined as a pair of adjacent parallel or antiparallel hydrogen-bonded beta-strands, packing with an alpha-helix shows a better correlation between the interaxial distance and log(V/nda) for the residues in the packing interface. This packing relationship is shown to be useful in the prediction of interaxial distances in alpha/beta units using the interacting residue information of equivalent alpha/beta units of homologous proteins. It is, therefore, of value in comparative modeling of protein structures.     

The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy

Cytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 A resolution, respectively. The enzyme forms an alpha/beta fold similar to bacterial cytidine deaminase, but with no similarity to the alpha/beta barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates.     

Pressure-induced transformation of alpha-helix to beta-sheet in the secondary structures of amyloid beta (1-40) peptide exacerbated by temperature

The effect of pressure on the conformational structure of amyloid beta (1-40) peptide (A beta(1-40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid A beta(1-40) from 1655 cm(-1) (alpha-helix) to 1647-1643 cm(-1) (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm(-1) assigned to beta-antiparallel sheet structure was also evident. Furthermore, the peak at 1540 cm(- 1) also shifted to 1527 (1529) cm(-1) in amide II band. The former was assigned to the combination of alpha-helix and random coil structures, and the latter was due to beta-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed A beta(1-40) samples were obtained from 33% to 22% for alpha-helix/random coil structures and from 47% to 57% for beta-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from alpha-helix to random coil and to beta- sheet structure. The structural transformation of the compressed A beta(1-40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of beta-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the beta-sheet structure transformed. The thermal-dependent transition temperatures of solid A beta(1-40) prepared by different pressures were near 55-60 degrees C.     

Chain-length dependence of alpha-helix to beta-sheet transition in polylysine: model of protein aggregation studied by temperature-tuned FTIR spectroscopy

The chain-length dependence of the alpha-helix to beta-sheet transition in poly(L-lysine) is studied by temperature-tuned FTIR spectroscopy. This study shows that heterogeneous samples of poly(L-lysine), comprising polypeptide chains with various lengths, undergo the alpha-beta transition at an intermediate temperature compared to homogeneous ingredients. This holds true as long as each individual fraction of the polypeptide is capable of adopting an antiparallel beta-sheet structure. The tendency is that the longer chain is, the lower the alpha-beta transition temperature is, which has been linked to the presence of distorted or solvated helices with turns or beta sheets in elongating chains of poly(L-lysine). As such helical structures are apparently conducive to the alpha-beta transition, this draws a comparison to the hypothesis of metastable protein conformational states being a common stage in amyloid-formation pathways. The antiparallel architecture of the beta sheet is likely to reflect the pretransition interhelical interactions in poly(L-lysine). Namely, the chains are arranged in an antiparallel manner because of energetically favored antiparallel pre-assembly of dipolar alpha helices.     

Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.     

Prediction of the tertiary structure of the alpha-subunit of tryptophan synthase

The tertiary structure of the alpha-subunit of tryptophan synthase was proposed using a combination of experimental data and computational methods. The vacuum-ultraviolet circular dichroism spectrum was used to assign the protein to the alpha/beta-class of supersecondary structures. The two-domain structure of the alpha-subunit (Miles et al.: Biochemistry 21:2586, 1982; Beasty and Matthews: Biochemistry 24:3547, 1985) eliminated consideration of a barrel structure and focused attention on a beta-sheet structure. An algorithm (Cohen et al.: Biochemistry 22:4894, 1983) was used to generate a secondary structure prediction that was consistent with the sequence data of the alpha-subunit from five species. Three potential secondary structures were then packed into tertiary structures using other algorithms. The assumption of nearest neighbors from second-site revertant data eliminated 97% of the possible tertiary structures; consideration of conserved hydrophobic packing regions on the beta-sheet eliminated all but one structure. The native structure is predicted to have a parallel beta-sheet flanked on both sides by alpha-helices, and is consistent with the available data on chemical cross-linking, chemical modification, and limited proteolysis. In addition, an active site region containing appropriate residues could be identified as well as an interface for beta 2-subunit association. The ability of experimental data to facilitate the prediction of protein structure is discussed.