Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study.

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis.

We have expanded our reference set of proteins used in the estimation of protein secondary structure by CD spectroscopy from 29 to 37 proteins by including 3 additional globular proteins with known X-ray structure and 5 denatured proteins. We have also modified the self-consistent method for analyzing protein CD spectra, SELCON3, by including a new selection criterion developed by W. C. Johnson, Jr. (Proteins Struct. Funct. Genet. 35, 307-312, 1999). The secondary structure corresponding to the denatured proteins was approximated to be 90% unordered, owing to the spectral similarity of the denatured proteins and unordered structures. We examined the thermal denaturation of ribonuclease T1 by CD using both the original and expanded sets of reference proteins and obtained more consistent results with the expanded set. The expanded set of reference proteins will be helpful for the determination of protein secondary structure from protein CD spectra with higher reliability, especially of proteins with significant unordered structure content and/or in the course of denaturation.

Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set.

We have expanded the reference set of proteins used in SELCON3 by including 11 additional proteins (selected from the reference sets of Yang and co-workers and Keiderling and co-workers). Depending on the wavelength range and whether or not denatured proteins are included in the reference set, five reference sets were constructed with the number of reference proteins varying from 29 to 48. The performance of three popular methods for estimating protein secondary structure fractions from CD spectra (implemented in software packages CONTIN, SELCON3, and CDSSTR) and a variant of CONTIN, CONTIN/LL, that incorporates the variable selection method in the locally linearized model in CONTIN, were examined using the five reference sets described here, and a 22-protein reference set. Secondary structure assignments from DSSP were used in the analysis. The performances of all three methods were comparable, in spite of the differences in the algorithms used in the three software packages. While CDSSTR performed the best with a smaller reference set and larger wavelength range, and CONTIN/LL performed the best with a larger reference set and smaller wavelength range, the performances for individual secondary structures were mixed. Analyzing protein CD spectra using all three methods should improve the reliability of predicted secondary structural fractions. The three programs are provided in CDPro software package and have been modified for easier use with the different reference sets described in this paper. CDPro software is available at the website: http://lamar.colostate.edu/ approximately sreeram/CDPro.

Molecular dynamics simulations of polypeptide conformations in water: A comparison of alpha, beta, and poly(pro)II conformations.

A significant fraction of the so-called "random coil" residues in globular proteins exists in the left-handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in alpha-helix, beta-strand, and left-handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the alpha-helix conformation and a relatively larger flexibility for the beta-strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of beta-strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (phi, psi) angles reflect a continuum of structures including both beta and P(II) conformations, but with a preference for local P(II) regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of beta and P(II) conformations. Such water bridges help stabilize the P(II) conformation relative to the beta conformation. Proteins 1999;36:400-406.

Tyrosine, phenylalanine, and disulfide contributions to the circular dichroism of proteins: circular dichroism spectra of wild-type and mutant bovine pancreatic trypsin inhibitor.

Improved descriptions of the lowest energy excited states of tyrosine and phenylalanine side chains have been developed in order to extend the capabilities of calculating the circular dichroism (CD) spectra of proteins. Four transitions (Lb, La, Bb, and Ba) for each of the side-chain chromophores were considered, and the transition monopole charges were obtained from a CNDO/S calculation on models representing the individual groups. Monopole charges at midpoints of the bonds, corresponding to the maximum transition charge densities in the Lb band, and monopole charges representing the vibronic coupling with the B transitions for the La transition were also included. The aromatic transitions were combined with the peptide transitions (npi, pi0pi n'pi, and pi+pi) and disulfide transitions (n1sigma and n4sigma) in the framework of the origin-independent matrix method to compute the CD spectra of different crystal forms and Y --> L and F --> L mutants of bovine pancreatic trypsin inhibitor (BPTI). The structures of the mutants were obtained by replacing the appropriate tyrosine or phenylalanine residue by leucine in the wild-type crystal structure. The CD calculations were performed on the energy-minimized structures. The CD spectrum calculated for the form II crystal structure of BPTI showed the best agreement with experiment. In the far UV, the calculated and experimental CD spectra agree to various extents for the wild-type and mutant BPTI. Among the mutants, the calculated CD spectra of Y4L, Y10L, Y23L, and F45L showed reasonable agreement with experiment, while those of Y21L and F22L, the two residues interacting with most aromatic groups, showed poor agreement. In the near UV, the negative bands predicted for the wild-type and mutant BPTI have much less intensity than observed experimentally.

Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy.

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.

Circular dichroism and electron microscopy of a core Y61F mutant of the F1 gene 5 single-stranded DNA-binding protein and theoretical analysis of CD spectra of four Tyr --> Phe substitutions.

A core Y61F mutant of the gene 5 single-stranded DNA-binding protein (g5p) of f1 bacterial virus aggregated when expressed from a plasmid, but, after refolding in vitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism (CD) titrations showed the same cooperative polynucleotide binding modes for Y61F and wild-type g5p. There are n = 4 and n congruent with 2.5 modes for binding to poly[d(A)] at low ionic strengths, but n = 4, n = 3, and n congruent with 2-2.5 modes for binding to fd single-stranded viral DNA (fd ssDNA), where n is the number of nucleotides occluded by each bound g5p monomer in a given mode. Y61F g5p has slightly reduced affinity in the n = 4 mode. Electron microscopy showed that Y61F g5p forms left-handed nucleoprotein superhelices indistinguishable from wild-type. Progression from binding to fd ssDNA in the n = 4 to n = 3 to n congruent with 2-2.5 mode is accompanied by an increase in the number of helical turns, an increase from (7.7 +/- 0.3) to (9.5 +/- 0.3) to ( approximately 10-13) g5p dimers per turn, and a decrease in the number of DNA nucleotides per turn. From CD spectra for four of five possible Y --> F g5p mutants, we infer that the fifth tyrosine, Tyr 56, contributes strongly to the CD. Retention of a strong 229 nm CD band in all mutants indicates that all retain elements of the native structure. Spectra of Y26F, Y34F, and Y61F g5p imply limited mobility of the replacement Phe. Comparison of measured with calculated CD spectra also suggests limited mobility for Tyr 26 and Tyr 34 in g5p in solution, and provides new information that the g5p structure in solution may be dominated by Tyr 41 rotamers differing from that stabilized in the crystal.

Synthesis and chiroptical properties of a novel C2-symmetric binaphthyl phosphortriamide ("chiral HMPA").

By use of an asymmetric Ullmann coupling involving chiral naphthalene oxazolines 1, the title compounds were prepared in good yields and with high diastereoselectivity. Hydrolysis of the binaphthyl oxazolines 2 led to the di-aldehydes 5, which were transformed into the azepine derivative, 6. The latter was treated with the appropriate phosphoryl halide to access the chiral HMPA systems 7 and 9. The CD spectra of the chiral azepine 6 and the chiral phosphoramides 7 and 9 were measured and showed a strong positive CD couplet near 225 nm, consistent with the P axial chirality (S configuration). Semi-empirical CNDO/S molecular orbital calculations of the CD spectrum of 6 satisfactorily reproduced the major features of the observed spectrum.

Protein secondary structure from circular dichroism spectroscopy. Combining variable selection principle and cluster analysis with neural network, ridge regression and self-consistent methods.

Different approaches to improve the analysis of protein secondary structure from circular dichroism spectra are compared. Grouping proteins based on the similarity of their circular dichroism spectra, using cluster analysis methods, was utilized as a new way of implementing variable selection. The performance of three basic methods (neural networks, ridge regression and singular value decomposition) was evaluated in combination with three approaches to improve the predictions; namely, variable selection, cluster analysis and the self-consistent method. Cluster analysis performed on the basis set proteins resulted in three clusters, subanalyses of which provide a new way of performing variable selection. The neural network with two hidden layers performed better than that with one hidden layer and was combined with variable selection. Inclusion of the variable selection principle improved the performance of all three basic methods. While the neural network method performed slightly better than the other two methods at the basic level, the inclusion of variable selection led to similar performance indices for all three methods.

Poly(pro)II helices in globular proteins: identification and circular dichroic analysis.

A method to identify poly(L-proline)-type (PII) conformation in crystal structures of globular proteins is presented. Short segments of PII structure were identified in globular protein structures, and these form a significant fraction of the residues which are not assigned to alpha-helix, beta-sheet, and beta-turns. The fractions of alpha-helix, beta-sheet, beta-turns, PII, and unordered, identified in conjunction with the Kabsch and Sander method [(1983) Biopolymers 22, 2577], were incorporated in the analysis of circular dichroism (CD) spectra of proteins. The separation of PII fraction from the fraction of residues not assigned to alpha-helix or beta-sheet or -turns resulted in a distinctive PII CD spectrum and an unusual CD spectrum corresponding to the residual unassigned structures. The quality of prediction of PII fraction from CD spectra of proteins was comparable to that of beta-sheet and -turns.

Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization.

It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (alpha-helix, 43%; beta-sheet, 14%; beta-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the lambda max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two- to threefold decrease in (kcat)app and an approximately two- to threefold increase in (Km)app, relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in (kcat)app and a two- to threefold increase in (Km)app, supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can synthesize the trimer GGA with an approximately 50-fold decrease in (kcat)app and a tenfold increase in (Km)app, relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in (kcat)app and an approximately 35-fold increase in (Km)app. Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Optical activity of hemoproteins in the Soret region. Circular dichroism of the heme undecapeptide of cytochrome c in aqueous solution.

Different possible mechanism for generation of optical activity of hemoproteins in the Soret region are reconsidered. The heme undecapeptide of cytochrome c does not contain aromatic amino acid residues, so its considerable optical activity cannot be due to coupling of heme pi pi * transitions with those of aromatic residues. CD data for the heme undecapeptide and for ferrimyoglobin and some of their complexes with small molecules are presented and critically compared. Symmetrically coordinated imidazole complexes show rotational strengths of the same magnitude as those of corresponding nonsymmetrically coordinated compounds. Inherent chirality in the bound heme is inferred to be a significant source of optical activity in the heme undecapeptide. Theoretical calculations based upon a molecular dynamics simulation support this proposal. Coupled oscillator interactions with the peptide pi pi * transitions and with the high-energy transitions in the peptide groups and thioether sulfurs, as modeled by polarizabilities, also make significant contributions. These same mechanisms must also be considered in hemoproteins in general.

A self-consistent method for the analysis of protein secondary structure from circular dichroism.

A self-consistent procedure for estimating the secondary structure content from circular dichroism spectra of proteins is presented. In this method the spectrum of the protein to be analyzed is included in the basis set and an initial guess is made for the unknown structure as a first approximation. The resulting matrix equation is solved using the singular value decomposition algorithm and the initial guess is replaced by the solution. The process is repeated until self-consistency is attained. The best features of the variable selection and the locally linearized methods are incorporated in this procedure. We have applied this method to examine the inconsistencies in the CD data, to compare the predictions with different ranges and resolutions of the CD data, and to compare different assignments of secondary structures from X-ray structure analyses in the context of secondary structure predictions. The results are compared using the root mean square differences and correlation coefficients. The results obtained are as good as or better than the previous analyses. For most of the proteins considered the self-consistent solutions obtained with different initial guesses were similar. We find the Kabsch and Sander protein crystal structure analysis to be most suitable for our prediction method.

Characterization of proline-containing right-handed alpha-helix by molecular dynamics studies.

Proline residues play a special role in shaping the secondary and tertiary structures of proteins. Many of these aspects have been studied in great detail. Current interest lies in elucidating the structure of right-handed alpha-helical fragments which contain proline in the middle of the helix. Such structures play an important role in membrane proteins and in the tight packing of globular proteins. Analysis of several crystal structures and energy minimization using flexible geometry have elucidated the nature of the bend produced by proline in the right-handed alpha-helical structure. Molecular dynamics (MD) simulation studies are ideally suited to characterize rigidity or flexibility in different parts of the molecule and can also give an idea of various conformations of the molecule which can exist at a given temperature. Hence, MD studies on Ace-(Ala)6-Pro-(Ala)3-NHMe have been carried out for 100 ps after equilibration and the resulting trajectories have been analyzed. Information regarding the average values, r.m.s. fluctuations of internal parameters and the time spent in different conformations are discussed. Energy minimization has been carried out on selected MD simulated points in order to analyze the characteristics of different conformations.