Beta-strand Structure Research Articles

Structural studies of bean pod mottle virus, capsid and RNA in crystal and solution states by laser Raman spectroscopy

Structures of protein and RNA components of bean pod mottle virus (BPMV) have been investigated by use of laser Raman spectroscopy. Raman spectra were collected from both aqueous solutions and single crystals of BPMV capsids (top component) and virions (middle and bottom components, which package, respectively, small and large RNA molecules). Analysis of the data permits the assignment of conformation-sensitive Raman bands to viral protein and RNA constituents and observation of structural similarities and differences between solution and crystalline states of BPMV components. The Raman results show that the protein subunits of the empty capsid contain between 45% and 55% beta-strand and beta-turn secondary structure, in agreement with the recently determined X-ray crystal structure, and that this total beta-strand content undergoes a small increase (approximately 5%) with packaging of RNA. The subunits are relatively deficient in alpha-helix secondary structure, estimated at less than 25%, and therefore must contain extensive amounts (greater than 20%) of loops and irregular chain conformations. The Raman spectra also show the following: (1) The molecular environments of as many as four tryptophan residues per subunit are altered upon packaging RNA, resulting in stronger 1N-H hydrogen bonding for two Trp residues and more hydrophobic environments for two indole rings. (2) Hydrogen-bonding states of the seven Tyr residues per subunit do not change detectably when RNA is packaged. At least five tyrosine OH groups are involved exclusively as strong hydrogen bond donors to protein acceptor groups, which suggests restricted access of solvent H2O molecules to these parahydroxyls.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational stability of P22 tailspike proteins carrying temperature-sensitive folding mutations

The thermostable tailspike endorhamnosidase of Salmonella phage P22 provides a model system for comparing the role of amino acid sequences in determining the intracellular folding pathway with their role in stabilizing the mature structural protein. Complete Raman band assignments are given here for the native form of the tailspike trimer in aqueous solution. Once correctly folded and assembled, the wild-type and two well-characterized mutant proteins, tsfIle258----Leu and tsfGly323----Asp, exhibit the same secondary structure in solution, consisting predominantly of beta-strand (56 +/- 5%) and turns (17 +/- 2%). Raman bands that are sensitive indicators of hydrogen-bonding interactions of tyrosine (phenolic OH) and tryptophan (indole NH) are unchanged between 30 and 80 degrees C in both wild type and tsf mutants. Similarly, Raman bands that are sensitive to changes in the hydrophobic environment of nonpolar side chains exhibit no significant temperature dependence in wild type and tsf mutants. In contrast, these conformational features are greatly altered by chemical denaturation of the tailspike with lithium halide and guanidine hydrochloride. In the chemically denatured tailspike, the beta-strand structure is substantially converted to irregular or "random coil" conformation. These findings confirm conclusions from physiological studies that the three-dimensional structures of the tsf mutants, once stabilized at permissive temperatures, are equivalent to the native structure of the wild type, and this structure is maintained at temperatures far above those that block the folding of the chain into the final native conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

The Bacillus thuringiensis delta-endotoxin. Evidence for a two domain structure of the minimal toxic fragment.

The conformational characteristics of the minimal toxic fragment of the delta-endotoxin from Bacillus thuringiensis berliner 1715 were examined by fluorescence and circular dichroism spectroscopy. This insecticidal protein, specifically toxic to lepidopteran species, was found to consist of two structural domains. Experimental evidence for this conclusion was provided by biphasic guanidine hydrochloride unfolding curves at different pH values and electrophoretic patterns of protease digests. Two stable fragments of comparable molecular weight were obtained using four different broad specificity proteolytic enzymes. A secondary structure model was constructed using seven B. thuringiensis toxin sequences. These toxins were selected on the basis of their limited sequence homology and represent all known insecticidal specificities. Despite this divergence, a consensus secondary structure pattern was obtained, confirming the structural homology among the toxins. The N-terminal halves of all toxins are predicted to be relatively rich in alpha-helix structure and the C-terminal parts to contain alternating beta-strand and coil structures. The latter seems characteristic for a beta-sheet conformation. Comparing this model to the unfolding data obtained by circular dichroism, whose far UV signal gives a measure of the alpha-helix content, allowed us to delineate the structural domains into the primary structure.

Fourier transform infrared spectroscopy for the characterization of a model peptide–DNA interaction

To better understand the structural basis of protein-DNA interactions, the conformational changes that accompany these interactions need to be described. In order to develop a methodological approach to this problem, Fourier transform infrared spectroscopy (FTIR) with derivative resolution enhancement has been used to identify conformational changes that occur when a 29-residue synthetic peptide binds nonspecifically to heterogeneous cellular DNA in aqueous solution. The peptide sequence was chosen de novo, in order to rationally design a peptide model that would allow the relationship between DNA binding and the stability of protein secondary structure to be studied. Peptide at a concentration of 100-200 microM produces 50% saturation of heterogeneous phage DNA sequences as well as of short synthetic oligonucleotides. FTIR spectra reveal significant changes in peptide and DNA upon binding. Second-derivative spectra resolve the amide I band of native peptide into components located at 1627 (beta-strand), 1658 (alpha-helix), and 1681 (turn or beta-strand) cm-1, with a distinct shoulder at 1647 cm-1 (disordered structure). Assignment of the 1681 cm-1 vibration to a turn conformation is supported by uv CD studies, which indicate significant amounts of turn structure in unbound peptide. Ultraviolet CD also confirms the existence of disordered and beta-strand regions in the free peptide. Upon interacting with DNA the band at 1681 cm-1 (turn) is no longer seen; a new band appears at 1675 cm-1; the 1627 cm-1 band (beta-strand) is considerably reduced in intensity; the position of the alpha-helical (1658 cm-1) component remains unchanged; the shoulder at 1647 cm-1 (disorder) disappears. The new vibration at 1675 cm-1 is characteristic of beta-strand structures. The asymmetric stretch (vAS) of the DNA phosphates shifts from 1223 (unbound) to 1229 cm-1 (bound); the relative intensities of vAS and the PO2- symmetric stretch (vS) are altered upon peptide binding.(ABSTRACT TRUNCATED AT 400 WORDS)

NMR studies of Arc repressor mutants: proton assignments, secondary structure, and long-range contacts for the thermostable proline-8----leucine variant of Arc.

Arc repressor is a 53-residue sequence-specific DNA binding protein. We report the assignment of the proton NMR spectrum and the secondary structure for the thermostable PL8 variant of Arc. This mutant, which differs from wild type by a Pro-8----Leu substitution, was chosen for study because its enhanced stability allows spectra to be acquired at elevated temperatures where spectral resolution is higher. The first five residues of the protein play important roles in DNA binding but appear to be disordered in solution. Residues 6-14 form the remaining part of the N-terminal DNA binding region of the protein and assume an antiparallel beta-conformation. This indicates that Arc is a member of a new class of DNA binding proteins. The observed interresidue nuclear Overhauser effects are consistent with a beta-strand, gamma-turn, beta-strand structure for the residue 6-14 region, although other structures are also consistent with the data. The remaining portion of the protein is predominantly alpha-helical. Residues 16-26 and 35-50 form amphipathic alpha-helices which may pack together in a four-helix bundle in the protein dimer.

Secondary structure prediction: combination of three different methods.

A combination of three complementary secondary structure prediction methods is presented. The methods used are the GOR III method, the Homologue method and a new method, the bit pattern method, which is based on hydrophilic/hydrophobic residue patterns. For this purpose a hydropathy scale was developed and is presented here. The combination algorithm (Combine method) was designed to take the best results of each method and use their differences in order to improve the prediction. The combination yields 65.5% correctly predicted residues in three states: alpha-helix (H), beta-strand (E) and aperiodic structure (C) which is an improvement ranging from 2.5 to 6.5% compared with the individual methods when tested with a 67-polypeptide chain database. Seventy-five per cent of the regular secondary structure (H and E) runs are correctly located and beta-sheet runs are much better located by the Combine method in comparison to the other methods.

Fourier transform infrared spectroscopic study of the structure and conformational changes of the human erythrocyte glucose transporter.

Fourier transform infrared spectroscopy has been used to study the secondary structure of the human erythrocyte glucose transporter after purification and reconstitution in erythrocyte lipids. The spectra indicate that the glucose transporter contains, in addition to the predominant alpha-helical structure, an appreciable amount of beta-structure and random coil conformation. A study of the time dependency of H-2H exchange revealed that more than 80% of the polypeptide backbone is readily accessible to the solvent. This result indicates that a portion of the intramembrane-spanning region of the membrane protein is exposed to the solvent, suggesting the existence of an intraprotein aqueous channel. The residual (10-20%) portion of the protein which exchanges slowly includes some alpha-helical structure, probably situated in a hydrophobic environment inside the membrane. The infrared spectra of transporter preparations were also examined after incubation with substrate and substrate analogues. Compared with the spectra recorded under conditions in which the "inward-facing" form predominates, a small but reproducible shift in the bands assigned to alpha-helical and beta-strand structures is observed after incubation with 4,6-O-ethylidene-D-glucose, which largely fixes the transporter in the "outward-facing" conformation. An increase of temperature, which is known to increase the proportion of transporter in the outward-facing conformation, results in a similar shift in this alpha-helical absorption band.

Design and characterization of peptides with amphiphilic beta-strand structures.

To extend our studies on peptides and proteins with amphiphilic secondary structures, a series of peptides designed to form amphiphilic beta-strand structures was designed, synthesized, and characterized by circular dichroism and infrared spectroscopy. Amphiphilic beta-strand conformations may be likely to appear in a variety of surface-active proteins, including apolipoprotein B and fibronectin. In a beta-strand conformation, the synthetic peptides will possess a hydrophobic face composed of valine side chains and a hydrophilic face composed of alternating acidic (glutamic acid) and basic (ornithine or lysine) residues. The peptides studied had a variety of chain lengths (5, 9, and 13 residues), and had the amino groups either free or protected with the trifluoroacetyl group. While the peptides did not possess a high potential for beta-sheet formation based on the Chou Fasman parameters, they possessed significant beta-sheet content, with up to 90% beta-sheet calculated for the 13-residue protected peptide. The driving force for beta-sheet formation is the potential amphiphilicity of this conformation. The beta-strand conformation of the 13-residue deprotected peptide was stable in 50% trifluoroethanol, 6 M guanidine hydrochloride, and octanol. The peptides are strongly self-associating in water, which would reduce the unfavorable contacts of the hydrophobic residues with water. It is clear that small peptides can be designed to form stable beta-strand conformations.

Trypanosome variant-specific glycoproteins: a polygene protein family with multiple folding patterns?

Infection with the African trypanosomes gives rise to relapsing waves of parasitemia in the host. A predominant population of trypanosomes is present in each wave, and such predominant populations are usually serologically distinct from each other. Trypanosomes are covered by an extramembranous, highly antigenic, variant-specific glycoprotein coat that is 15 nm thick. The primary structure of a large portion of the glycoprotein molecule is different in the predominant trypanosome populations of each parasitemic wave. Analysis of the secondary structure potential of five full-length and five partial amino acid sequences of variant-specific glycoproteins from members of the Trypanosoma brucei complex has been carried out. The potentials for alpha-helix, beta-turns, and beta-strand structure have been calculated. A high degree of alpha-helical structure potential is present in all the full-length or partial sequences examined. There is conservation of secondary structure potential in the COOH-terminal 100 amino acids, where both partial and complete conservation of primary amino acid sequence exists. The NH2-terminal regions are rich in alpha-helix potential. However, over large stretches of the middle of the VSG molecules there is wide diversity of secondary structure potential. This suggests that tertiary folding structures may also be different in this region. If these predictions are true, different regions of the variant-specific glycoprotein could be exposed to the solvent in different variant-specific trypanosome serotypes. The implication is that antigenic variation is mediated by a polygene family of glycoproteins containing highly polymorphic regions. These could fold differently and expose different surface regions of the protein to the solvent. This device might reduce immune crossreactivity among members of the variant-specific glycoprotein family.

PREDICTED SECONDARY STRUCTURES OF FOUR PENICILLIN BETA‐LACTAMASES AND A COMPARISON WITH TWO LYSOZYMES

We have predicted the secondary structures of four beta-lactamases (Bacillus cereus, Bacillus licheniformis, Staphylococcus aureus, and Escherichia coli R-TEM) by the statistical method of Chou & Fasman as well as by the information theory method of Garnier et al. The secondary structures of all four beta-lactamases are of the alpha/beta type (Levitt & Chothia's nomenclature), with helices at N- and C-termini. There are about eight short regions each of alpha-helical (30--50%) and beta-strand (10--20%) structure separated by about 20 reverse turns. The conformation of the Gram-positive and Gram-negative beta-lactamases are generally similar although a few differences are predicted between the S.aureus and E.coli structures. Surprisingly, the two bacilli structures differ significantly in three short regions. In all four enzymes the region near the catalytically-implicated tyrosine has similar secondary structure. The secondary structure of hen egg white lysozyme, a penicillin-binding enzyme, as well as T4 phage lysozyme, has similarities to the N-terminal half of the penicillin-destroying beta-lactamases.