Analysis of the segmental stability of helical peptides by isotope-edited infrared spectroscopy.

Isotope-edited infrared spectroscopy has the ability to probe the segmental properties of long biopolymers. In this work, we have compared the infrared spectra of a model helical peptide ((12)C) Ac-W-(E-A-A-A-R)(6)-A-NH(2), described originally by Merutka et al. (Biochemistry 1991;30:4245-4248) and three derivatives that are (13)C labeled at the backbone carbonyl of alanines. The locations of six isotopically labeled alanines are at the N-terminal, C-terminal, and the middle two repeating units of the peptide. Variation in temperature from 1 degrees to 91 degrees C transformed the peptides from predominantly helical to predominantly disordered state. Amplitude and position of the infrared amide I' absorption bands from (12)C- and (13)C-labeled segments provided information about the helical content. Temperature dependence of infrared spectra was used to estimate segmental stability. As a control measure of overall peptide stability and helicity (independent of labeling), the temperature dependence of circular dichroism spectra in the far-UV range at identical conditions (temperature and solvent) as infrared spectra was measured. The results indicate that the central quarter of the 32 amino acids helix has the maximal helicity and stability. The midpoint of the melting curve of the central quarter of the helix is 5.4 +/- 0.8 degrees C higher than that of the termini. The N-terminal third of the helix is more helical and is 2.0 +/- 1.4 degrees C more stable than the C-terminus.

Expression and properties of bacteriophage T4 gene product 11.

A plasmid vector for expression of bacteriophage T4 gene product 11 (gp11) in E. coli cells has been constructed. Gp11 is a baseplate protein that connects short tail fibers providing irreversible adsorption of the virus on a cell. A method based on chromatography on hydroxyapatite has been developed for purification of recombinant gp11. The protein is active in an in vitro complementation assay and transforms defective phage particles lacking gp11 into infective ones. Gel filtration data suggest that the biologically active protein is a trimer. According to CD spectroscopy and sequence analysis data, the polypeptide chain of gp11 contains not less than 20% alpha-helical segments, about 30% beta-structure, and belongs to the class of alpha/beta structural proteins.

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.

Structure and folding of bacteriophage T4 gene product 9 triggering infection. I. Production and properties of recombinant protein.

Gene product 9 (gp9, 288 amino acid residues per monomer, molecular weight 30.7 kD) of bacteriophage T4 triggers the baseplate reorganization and the sheath contraction after interaction of the long tail fibers with the receptors of the bacterial cell. In this work we have produced the recombinant protein and determined that gp9 is a stable homotrimer and active in in vitro complementation assay completing the defective phage particles which lack gp9. According to CD-spectroscopy data, the gp9 polypeptide chain contains 65-73% beta-structure and 11-16% alpha-helical segments, this being in good agreement with secondary structure prediction results. Additionally, we have constructed a set of plasmid vectors for expression of gp9 deletion mutants. The fragments with consecutive truncations of the N-terminus of the molecule, as well as the full-length protein, are trimers resistant to SDS treatment and decrease infective phage particle formation in in vitro complementation assay with native gp9. The deletion of the molecule C-terminal region results in failure of trimerization and decreases the stability of the protein.

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.

Divalent cations stabilize the alpha 1 beta 1 integrin I domain.

Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.

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.

Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.

The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.

Thermodynamic study of the apomyoglobin structure.

Sperm whale apomyoglobin has been studied thermodynamically in solutions with different pH and temperature by scanning microcalorimetry, viscosimetry, nuclear magnetic resonance and circular dichroism spectrometry, and by electrometric and calorimetric titration. It has been shown that apomyoglobin in solutions with pH close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is maximally stable at about 30 degrees C and breaks down reversibly both upon heating or cooling from this temperature. The process of breakdown of this structure is highly co-operative and can be regarded as a transition between two macroscopic states of protein, the native and denatured states. In contrast to the native state, which is specified by definite values of compactness and ellipticity, the compactness and ellipticity of the denatured state of apomyoglobin depend strongly on pH; with a decrease of pH below 4.0, these parameters gradually approach the values of the random coil.

Identification and isolation of vinculin from platelets.

A vinculin-like protein was identified in chicken as well as in bovine platelets by ELISA competitive binding assay using antibodies against vinculin from chicken gizzard. By a modified procedure (J. Biol. Chem. (1980) 255, 1194-1199) we succeeded in isolating bovine platelet vinculin to apparent homogeneity. The structural identity of platelet and chicken gizzard vinculin was demonstrated by circular dichroism analysis. It was also shown that platelet vinculin induces a significant decrease in the low shear viscosity of F-actin. Vinculin, in all probability, plays an important role in the organization of actin filaments in platelets, especially in the linkages of microfilaments to the membrane.

Optical characteristics of all individual proteins from the small subunit of Escherichia coli ribosomes.

The procedure of isolation and renaturation of all ribosomal proteins from the 30-S subunit of Escherichia coli ribosomes is described. Absorption spectra of these proteins in the near-ultraviolet region have been measured and molar absorption coefficients have been determined on the basis of nitrogen content. Molar absorption coefficients have been calculated for 20 proteins with a known amino acid sequence and the calculated values have been compared with the experimentally determined ones. The absorption spectra obtained allow an easy, precise and highly reproducible spectrophotometric determination of the concentration of individual ribosomal proteins. Circular dichroic spectra of 21 individual proteins from the 30-S subunit of E. coli ribosomes were measured in the range 184-310 nm. The secondary structure of the proteins studied was calculated from the spectra in the range 190-240 nm. Almost all proteins (except proteins S12, S17, S18 and S19) are characterized by a high content of secondary structure. Circular dichroic spectra in the near-ultraviolet region (240-310 nm) indicate that the side groups of aromatic amino acids are fixed in the tertiary structure of the proteins studied. Some internal characteristics (independent of the measurement conditions) of the circular dichroic spectrum in the far-ultraviolet region were proposed as a measure of the resemblance to the native state of ribosomal proteins; these characteristics may be useful for comparison of protein preparations obtained by different methods in different laboratories.

Lack of gross protein structure changes in the working cycle of (Na+, K+)-dependent adenosinetriphosphatase. Evidence from infrared and intrinsic fluorescence spectroscopy data.

Infrared and tryptophan fluorescence spectra of practically all sufficiently stable functional complexes of a highly purified preparation of membrane-bound (Na+, K+)-dependent ATPase have been measured. The formation of any functional complex was not accompanied by any considerable change of either shape or position of the tryptophan fluorescence spectrum. Only in the presence of adenine nucleotides was there a small decrease of fluorescence intensity (by 5-8%), which apparently results from a change of the sample light scattering. Analysis of the results obtained leads to the conclusion that the environment of no more than one or a few tryptophan residues may differ in all the (Na+, K+)-ATPase complexes studies. A comparison of infrared protein spectra in the region of amide I band showed that at any wavenumber the differences between them did not exceed 3% of the maximum absorption. This means that no more than 3% of protein peptide groups can change their conformation upon transition between the enzyme functional states. These results, obtained by two independent techniques, allow us to conclude that even if changes of the internal protein structure occur during the working cycle of this transport system, if they have an extremely local character.

Cleavage of elongation factor G into compact domains.

A limited trypsinolysis of native elongation factor G results in the formation of two large fragments resistant to further proteolysis. The fragments were isolated in homogeneous state in conditions when their native structure is retained. According to circular dichroism and scanning calorimetry data the formed fragments retain the stability and compact structure that they had in the whole protein.