Individual Secondary Structural Elements Research Articles

Nonnative contact effects in protein folding.

A comprehensive understanding of protein folding includes the knowledge of the formation of individual secondary structures, tertiary structure, and the effects of non-native contacts on these folding events. The measurement of these microscopic events has been posing challenges for experiment and molecular simulation. In this work, we performed enhanced sampling MD simulations for three proteins (NTL9, NuG2b, and CspA) and analyzed minimum free energy paths on multi-dimensional free energy landscapes to explore the underlying folding mechanisms. Consistencies can be seen between the present simulations and the existing experiments as well as other MD simulations. Quantitative analysis reveals the nucleation-condensation folding mechanism indicating the concurrent build-up of secondary and tertiary structures for the three proteins and gives the detailed formation sequence of individual native secondary structure elements. More importantly, nonnative contacts are generally observed among the proteins, creating a nonnative environment to affect the folding of individual secondary structure elements. A general tendency is that the secondary structure element(s) where the maximal nonnative contacts are observed have the largest formation free-energy barrier(s), corresponding to the rate-limiting step(s) of the folding for proteins that follow the nucleation-condensation mechanism. In summary, while native contacts determine the folding mechanism and pathway, non-native contacts play an important role in determining the protein folding thermodynamics by influencing the free energies of individual secondary structure element formation.

Structural and thermal stability of β-lactoglobulin as a result of interacting with sugar beet pectin.

Changes in the structural and thermal stability of β-lactoglobulin (β-LG) induced by interacting with sugar beet pectin (SBP) have been studied by circular dichroism (CD), Fourier transform infrared, and steady-state as well as time-resolved fluorescence spectroscopic techniques. It has been demonstrated that SBP not only is capable of binding to native β-LG but also causes a significant loss in antiparallel β-sheet, ∼10%, accompanied by an increase in either random coil or turn structures. In addition, the interaction also disrupted the environments of all aromatic residues including Trp, Phe, and Tyr of β-LG as evidenced by near-UV CD and fluorescence. When preheated β-LG was combined with SBP, the secondary structure of β-LG was partially recovered, ∼4% gain in antiparallel β-sheet, and Trp19 fluorescence was recovered slightly. Although forming complexes with SBP did not significantly impact the thermal stability of individual secondary structural elements of β-LG, the environment of Trp19 was protected considerably.

Examining the Flexibility of Tim Barrel Proteins Based on their Structural Topology

The eight-fold beta/alpha barrel-like fold, first seen in triose-phosphate isomerase (TIM), is a common and versatile fold for proteins. Proteins in this classification have diverse enzymatic functions spanning five of six Enzyme Commission classes. Studies have characterized the sequence, structure, localized dynamics and electrostatics of TIM proteins, but the flexibility of the fold is understudied. We use coarse-grained and all-atom normal modes analysis to look at the principle TIM beta/alpha barrel secondary structure elements of five structures from distinct families with low sequence similarity between 23 and 38.5 % and low structure similarity. We report that the eight core beta strands behave independently of the eight flanking alpha helices. The strands and helices repeat in tandem at the sequence level, yet the strands form a core that moves minimally while the helices display greater relative mobility in all of the structures. This core formed by the beta-strands is rigid in all the structures. This is despite the varying loop lengths between them and the presence of accessory secondary structures in some cases. We found that the atomic fluctuations of each of the beta-strands have a high correlation with its immediate beta-strand neighbour in the core. Compared to the beta strands, the alpha helices have less correlation with each other and almost none with the strands. We also defined rotation and translation vectors to decompose the type of movements to which the normal modes of these TIMs correspond. We relate differences in the mobility of the individual secondary structure elements to the differences in function and structure of the five different enzymes.

Finding rigid bodies in protein structures: Application to flexible fitting into cryoEM maps

We present RIBFIND, a method for detecting flexibility in protein structures via the clustering of secondary structural elements (SSEs) into rigid bodies. To test the usefulness of the method in refining atomic structures within cryoEM density we incorporated it into our flexible fitting protocol (Flex-EM). Our benchmark includes 13 pairs of protein structures in two conformations each, one of which is represented by a corresponding cryoEM map. Refining the structures in simulated and experimental maps at the 5-15Å resolution range using rigid bodies identified by RIBFIND shows a significant improvement over using individual SSEs as rigid bodies. For the 15Å resolution simulated maps, using RIBFIND-based rigid bodies improves the initial fits by 40.64% on average, as compared to 26.52% when using individual SSEs. Furthermore, for some test cases we show that at the sub-nanometer resolution range the fits can be further improved by applying a two-stage refinement protocol (using RIBFIND-based refinement followed by an SSE-based refinement). The method is stand-alone and could serve as a general interactive tool for guiding flexible fitting into EM maps.

2D Correlation Deep UV Resonance Raman Spectroscopy of Early Events of Lysozyme Fibrillation: Kinetic Mechanism and Potential Interpretation Pitfalls

The early stages of hen egg white lysozyme (HEWL) fibrillation were quantitatively characterized by two-dimensional correlation deep UV resonance Raman spectroscopy (2D-DUVRR) in terms of the sequential order of events and their characteristic times. The evolution of individual secondary structural elements was established through the correlation between Amide I, Amide III, and Calpha-H bending Raman bands. The temporal order of tertiary and individual secondary structural changes was probed through the cross-correlation of phenylalanine and Amide Raman bands. Both the sequential order and the characteristic times of tertiary and secondary structural changes allowed for reconstructing the molecular mechanism of lysozyme structural changes at the early stages of fibrillation. The 2D-DUVRR analysis of our data indicated that melting of the alpha-helix happened after the formation of the disordered structure, which was termed as apparent inverse order of secondary structural changes. We demonstrated that this apparent inverse order of events is typical for all chemical reactions involving the formation of intermediate(s), which may lead to the serious misinterpretation of 2D correlation results. We proposed a new simulation-aided approach for reconstructing and quantitatively characterizing the reaction mechanism of a (bio)chemical reaction that accounts for the apparent inverse order of events.

Crystal Structure of Human Cytochrome P450 2D6

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

Using the example of sodium/proton antiporter from Escherichia coli NhaA, we review the capabilities of single-molecule atomic force microscopy and force spectroscopy to observe structural and functional insights of a membrane protein, which are not attainable by other traditional methods. While atomic force microscopy provides high-resolution topographs of single membrane proteins, their oligomeric state and assembly, single-molecule force spectroscopy experiments detect molecular interactions of the protein. The sensitivity of this method makes it possible to detect and locate interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within them. Controlled refolding experiments using single-molecule force spectroscopy observed individual secondary structure segments folding into the functional protein. Various folding pathways of NhaA were detected, each one exhibiting a certain probability to be taken. Time-lapse refolding experiments enabled determining the folding kinetics and hierarchy of individual secondary structural elements. Recent examples detected and located the ligand binding of an antiporter. Similarly, inhibitor binding and location can be detected which in future may guide towards comparative studies of agonist and antagonist altering the functional state of a membrane protein. We review current and future potentials of these approaches to characterize the action of pharmacological molecules on the antiporter function.

Resonance Assignments and Secondary Structure Analysis of E. coli Thioredoxin by Magic Angle Spinning Solid-State NMR Spectroscopy

De novo site-specific 13C and 15N backbone and sidechain resonance assignments are presented for uniformly enriched E. coli thioredoxin, established using two-dimensional homo- and heteronuclear solid-state magic angle spinning NMR correlation spectroscopy. Backbone dihedral angles and secondary structure were derived from the statistical analysis of the secondary chemical shifts, and are in good agreement with solution values for the intact full-length thioredoxin, with the exception of a small number of residues located at the termini of the individual secondary structure elements. A large number of cross-peaks observed in the DARR spectra with long mixing times correspond to the pairs of carbon atoms separated by 4-6 angstroms, suggesting that DARR could be efficiently employed for observation of medium- and long-range correlations. The 108 amino acid residue E. coli thioredoxin is the largest uniformly enriched protein assigned to this degree of completeness by solid-state NMR spectroscopy to date. It is anticipated that with a combination of two-dimensional correlation experiments and high magnetic fields, resonance assignments and secondary structure can be generally derived for other noncrystalline proteins.

Solution Structure of the Tctex1 Dimer Reveals a Mechanism for Dynein-Cargo Interactions

Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.

Methionine Adenosyltransferase α-Helix Structure Unfolds at Lower Temperatures than β-Sheet: A 2D-IR Study

Two-dimensional infrared spectroscopy has been used to characterize rat liver methionine adenosyltransferase and the events taking place during its thermal unfolding. Secondary structure data have been obtained for the native recombinant enzyme by fitting the amide I band of infrared spectra. Thermal denaturation studies allow the identification of events associated with individual secondary-structure elements during temperature-induced unfolding. They are correlated to the changes observed in enzyme activity and intrinsic fluorescence. In all cases, thermal denaturation proved to be an irreversible process, with a T m of 47–51°C. Thermal profiles and two-dimensional infrared spectroscopy show that unfolding starts with α-helical segments and turns, located in the outer part of the protein, whereas extended structure, associated with subunit contacts, unfolds at higher temperatures. The data indicate a good correlation between the denaturation profiles obtained from activity measurements, fluorescence spectroscopy, and the behavior of the infrared bands. A study of the sequence of events that takes place is discussed in light of the previous knowledge on methionine adenosyltransferase structure and oligomerization pathway.

The relationship between third-codon position nucleotide content, codon bias, mRNA secondary structure and gene expression in the drosophilid alcohol dehydrogenase genes Adh and Adhr.

To gain insights into the relationship between codon bias, mRNA secondary structure, third-codon position nucleotide distribution, and gene expression, we predicted secondary structures in two related drosophilid genes, Adh and Adhr, which differ in degree of codon bias and level of gene expression. Individual structural elements (helices) were inferred using the comparative method. For each gene, four types of randomization simulations were performed to maintain/remove codon bias and/or to maintain or alter third-codon position nucleotide composition (N3). In the weakly expressed, weakly biased gene Adhr, the potential for secondary structure formation was found to be much stronger than in the highly expressed, highly biased gene Adh. This is consistent with the observation of approximately equal G and C percentages in Adhr ( approximately 31% across species), whereas in Adh the N3 distribution is shifted toward C (42% across species). Perturbing the N3 distribution to approximately equal amounts of A, G, C, and T increases the potential for secondary structure formation in Adh, but decreases it in Adhr. On the other hand, simulations that reduce codon bias without changing N3 content indicate that codon bias per se has only a weak effect on the formation of secondary structures. These results suggest that, for these two drosophilid genes, secondary structure is a relatively independent, negative regulator of gene expression. Whereas the degree of codon bias is positively correlated with level of gene expression, strong individual secondary structural elements may be selected for to retard mRNA translation and to decrease gene expression.

Interaction of hepatitis A virus (HAV) precursor proteins 3AB and 3ABC with the 5′ and 3′ termini of the HAV RNA

RNA secondary structures within the terminal nontranslated regions of entero- and rhinoviral genomes interact specifically with viral nonstructural proteins and are required in cis for viral RNA replication. Here we show that recombinant hepatitis A virus (HAV) polypeptide 3ABC specifically interacts in vitro with secondary RNA structures formed at both the 5′ and 3′ terminus of the viral genome. Similar to protein 3AB, HAV 3ABC bound to the 3′ terminal RNA structure which did not interact with the mature proteinase 3C. In contrast to 3AB, 3ABC interacted with RNA stem-loop IIa and combinations of individual secondary structure elements of the 5′ noncoding region. RNA binding of the precursor polypeptide 3ABC was 50 times stronger than that of 3AB and 3C, implicating a specific role of this stable processing intermediate in viral genome replication.

Molecular dynamics simulations of N-terminal peptides from a nucleotide binding protein.

Molecular dynamics (MD) simulations of N-terminal peptides from lactate dehydrogenase (LDH) with increasing length and individual secondary structure elements were used to study their stability in relation to folding. Ten simulations of 1-2 ns of different peptides in water starting from the coordinates of the crystal structure were performed. The stability of the peptides was compared qualitatively by analyzing the root mean square deviation (RMSD) from the crystal structure, radius of gyration, secondary and tertiary structure, and solvent accessible surface area. In agreement with earlier MD studies, relatively short (< 15 amino acids) peptides containing individual secondary structure elements were generally found to be unstable; the hydrophobic alpha 1-helix of the nucleotide binding fold displayed a significantly higher stability, however. Our simulations further showed that the first beta alpha beta supersecondary unit of the characteristic dinucleotide binding fold (Rossmann fold) of LDH is somewhat more stable than other units of similar length and that the alpha 2-helix, which unfolds by itself, is stabilized by binding to this unit. This finding suggests that the first beta alpha beta unit could function as an N-terminal folding nucleus, upon which the remainder of the polypeptide chain can be assembled. Indeed, simulations with longer units (beta-alpha-beta-alpha and beta-alpha-beta-alpha beta-beta) showed that all structural elements of these units are rather stable. The outcome of our studies is in line with suggestions that folding of the N-terminal portion of LDH in vivo can be a cotranslational process that takes place during the ribosomal peptide synthesis.

A predicted consensus structure for the protein kinase C2 homology (C2H) domain, the repeating unit of synaptotagmin.

A secondary structure has been predicted for the protein kinase C2 regulatory domain found in homologous form in synaptotagmin, some phospholipases, and some GTP activated proteins. The proposed structure is built from seven consecutive beta strands followed by a terminal alpha helix. Considerations of overall surface exposure of individual secondary structural elements suggest that these are packed into a 2-sheet beta sandwich structure, with one of only three of the many possible folds being preferred.

Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability.

To investigate the relation between protein stability and the predicted stabilities of individual secondary structural elements, residue Pro86 in an alpha-helix in phage T4 lysozyme was replaced by ten different amino acids. The x-ray crystal structures of seven of the mutant lysozymes were determined at high resolution. In each case, replacement of the proline resulted in the formation of an extended alpha-helix. This involves a large conformational change in residues 81 to 83 and smaller shifts that extend 20 angstroms across the protein surface. Unexpectedly, all ten amino acid substitutions marginally reduce protein thermostability. This insensitivity of stability to the amino acid at position 86 is not simply explained by statistical and thermodynamic criteria for helical propensity. The observed conformational changes illustrate a general mechanism by which proteins can tolerate mutations.