Mechanism of selection of side-chain rotamers in α-helices

In this study, a possible mechanism of selection of side-chain rotamers based on the rotamer distributions in known coiled-coil proteins is suggested. According to this mechanism, interhelical hydrophobic, polar, and packing interactions bring alpha-helices closer to each other and this effect squeezes side chains out of the helix-helix interface. As a result, in dimeric coiled coils and long alpha-alpha-hairpins where alpha-helices are packed in a face-to-face manner, most side chains occupying the a-positions have t-rotamers and those in the d-positions g(-)-rotamers. In tetramers, where alpha-helices are packed side-by-side, most side chains in the a-positions adopt g(-)-rotamers and those in the d-positions t-rotamers.     

Side-chain rotamers in protein α-α hairpins and a mechanism of their selection

Several regularities were observed for the distribution of side-chain rotamers in α-α hairpins of globular proteins. In left-turned α-α hairpins, most side chains adopt t rotamers in d-positions and g^? rotamers in g-positions. In right-turned α-α hairpins, most side-chains adopt g^? rotamers in a-positions and t rotamers in e-positions. Analysis of these regularities suggested that selection of the side-chain conformation in α-α hairpins depends on two main factors, the mode of α-helix packing and the positions of side chains in α-helices. The regularities were explained by the squeezing mechanism: interhelical interactions bring the α-helices close together so that the side chains are squeezed out of the helix-helix interface and adopt unique conformations.     

Packing scheme of α-helices in the histone core of the nucleosome

A nucleosome histone core model is presented which is compatible with experimental data. The model consists of 28 α-helices located as predicted by others^1–4. The histone wheel resembles the one proposed by Klug et al.⁵ Most of the helices are packed nearly parallel to the DNA superhelical axis, forming a bandoleer-like structure. About 10 to 20% of the nucleosomal phosphates may be neutralized by positively charged residues in the α-helices. Disregarding the charge of the NH₂-terminals, this is sifficient for the thermodynamic stability of the nucleosome under physiological conditions. The electrostatic charge on the protein surface is assumed to be relatively fixed due to the participation of the corresponding side chains to the hydrophobically packed helices. Thus, DNA wrapping appears to be determined by the core histones not by the histone NH₂-terminals.     

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts     

Role of the N-terminal hydrophobic residues of DGKε in targeting the endoplasmic reticulum

The endoplasmic reticulum (ER), comprised of an interconnected membrane network, is a site of phospholipid and protein synthesis. The diacylglycerol kinase (DGK) enzyme family catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Both of these lipids are known not only to serve as second messengers but also to represent intermediate precursors of lipids of various kinds. The DGK family is targeted to distinct subcellular sites in cDNA-transfected and native cells. Of DGKs, DGKε localizes primarily to the ER, suggesting that this isozyme plays a role in this organelle. Using experiments with various deletion and substitution mutants, this study examined the molecular mechanism of how DGKε is targeted to the ER. Results demonstrate that the N-terminal hydrophobic sequence 20–40 plays a necessary role in targeting of DGKε to the ER. This hydrophobic amino acid segment is predicted to adopt an α-helix structure, in which Leu22, L25, and L29 are present in mutual proximity, forming a hydrophobic patch. When these hydrophobic Leu residues were replaced with hydrophilic amino acid Gln, the mutant fragment designated DGKε (20–40/L22Q,L25Q,L29Q) exhibits diffuse distribution in the cytoplasm. Moreover, full-length DGKε containing these substitutions, DGKε (L22Q,L25Q,L29Q), is shown to distribute diffusely in the cytoplasm. These results indicate that the N-terminal hydrophobic residues play a key role in DGKε targeting to the ER membrane. Functionally, knockdown or deletion of DGKε affects the unfolding protein response pathways, thereby rendering the cells susceptible to apoptosis, to some degree, under ER stress conditions.     

Role of α-Factor and the Mfα1 α-Factor Precursor in Mating in Yeast

The peptide pheromones secreted by a and α cells (called a-factor and α-factor, respectively) are each encoded by two structural genes. For strains of either mating type, addition of exogenous pheromone does not alleviate the mating defect of mutants with disruptions of both structural genes. In addition, a particular insertion mutation in the major α-factor structural gene (MFα1) that should result in an altered product inhibits α mating. These results suggested that the pheromone precursors (the MFα1 pro region in particular) might play a second role in mating separate from the role of pheromone production. To analyze the role of α-factor and the MFα1 precursor in α mating, we have constructed two classes of mutants. The mating defects of mutants that should produce the MFα1 pro region peptide but no α-factor could not be alleviated by addition of exogenous α-factor in crosses to a wild-type a strain, indicating that the previous results were not due to an inability of the disruption mutants to produce the pro region peptide. Mutants able to produce α-factor, but with a variety of alterations in MFα1 precursor structure, mated at levels proportional to the levels of α-factor produced, suggesting that the only role of the α-factor precursor in mating is to produce α-factor. Both of these results argue against a role for the MFα1 pro region separate from its role in α-factor production. We also describe results that show that in vivo production of α-factor'' (the form of α-factor encoded by one of the two α-factor repeats of MFα2) is equivalent to the major form of α-factor for induction of all responses necessary for mating. We discuss the implications of these results on the role of the pheromones in mating.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Transmembrane orientation of α-helices in the thylakoid membrane and in the light-harvesting complex. A polarized infrared spectroscopy study

The structure and orientation of the major protein constituent of photosynthetic membranes in green plants, the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) have been investigated by ultraviolet circular dichroism (CD) and polarized infrared spectroscopies. The isolated purified LHC has been reconstituted into phosphatidylcholine vesicles and has been compared to the pea thylakoid membrane. The native orientation of the pigments in the LHC reconstituted in vesicles was characterized by monitoring the low-temperature polarized absorption and fluorescence spectra of reconstituted membranes. Conformational analysis of thylakoid and LHC indicate that a large proportion of the thylakoid protein is in the α-helical structure (56 ± 4%), while the LHC is for 44 ± 7% α-helical. By measuring the infrared dichroism of the amide absorption bands of air-dried oriented multilayers of thylakoids and LHC reconstituted in vesicles, we have estimated the degree of orientation of the α-helical chains with respect to the membrane normal. Infrared dichroism data demonstrate that transmembrane α-helices are present in both thylakoid and LHC with the α-helix axes tilted at less than 30° in LHC and 40° in thylakoid with respect to the membrane normal. In thylakoids, an orientation of the polar C=O ester groups of the lipids parallel to the membrane plane is detected. Our results are consistent with the existence of 3–5 transmembrane α-helical segments in the LHC molecules.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of αA- and αB-crystallins

Earlier studies have shown significant loss of chaperone activity in α-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of α-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in α-crystallin have been identified as the major glycation sites: K11, K78, and K166 in αA-crystallin and K90, K92, and K166 in αB-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of αA showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to αA-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of αB showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. αA-K11T and αB-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.     

Characterization and sequence prediction of structural variations in α-helix

Background

Centralised resources such as GenBank and UniProt are perfect examples of the major international efforts that have been made to integrate and share biological information. However, additional data that adds value to these resources needs a simple and rapid route to public access. The Distributed Annotation System (DAS) provides an adequate environment to integrate genomic and proteomic information from multiple sources, making this information accessible to the community. DAS offers a way to distribute and access information but it does not provide domain experts with the mechanisms to participate in the curation process of the available biological entities and their annotations.

Results

We designed and developed a Collaborative Annotation System for proteins called DAS Writeback. DAS writeback is a protocol extension of DAS to provide the functionalities of adding, editing and deleting annotations. We implemented this new specification as extensions of both a DAS server and a DAS client. The architecture was designed with the involvement of the DAS community and it was improved after performing usability experiments emulating a real annotation task.

Conclusions

We demonstrate that DAS Writeback is effective, usable and will provide the appropriate environment for the creation and evolution of community protein annotation.     

Mitochondrial Ageing and the Beneficial Role of α-Lipoic Acid

Oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the ageing process. Mitochondria are known to be a rich source for the production of free radicals and, consequently, mitochondrial components are susceptible to lipid peroxidation (LPO) that decreases respiratory activity. In the present investigation, we have evaluated mitochondrial LPO, 8-oxo-dG, oxidized glutathione, reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and electron transport chain (ETC) complex activities in the brain of young versus aged rats. In aged rats, the contents of LPO, oxidized glutathione and 8-oxo-dG were high whereas reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities were found to be low. Lipoic acid administration to aged rats reduced the levels of mitochondrial LPO, 8-oxo-dG and oxidized glutathione and enhanced reduced glutathione, ATP, lipoic acid and ETC complex activities. In young rats lipoic acid administration showed only minimal lowering the levels of LPO, 8-oxo-dG and oxidized glutathione and slight increase in the levels of reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities. These findings suggest that the dithiol, lipoic acid, provides protection against age-related oxidative damage in the mitochondria of aged rats.     

Signature of n→π* interactions in α-helices

The oxygen of a peptide bond has two lone pairs of electrons. One of these lone pairs is poised to interact with the electron-deficient carbon of the subsequent peptide bond in the chain. Any partial covalency that results from this n→π* interaction should induce pyramidalization of the carbon (C'(i)) toward the oxygen (O(i-1)). We searched for such pyramidalization in 14 peptides that contain both α- and β-amino acid residues and that assume a helical structure. We found that the α-amino acid residues, which adopt the main chain dihedral angles of an α-helix, display dramatic pyramidalization but the β-amino acid residues do not. Thus, we conclude that O(i-1) and C'(i) are linked by a partial covalent bond in α-helices. This finding has important ramifications for the folding and conformational stability of α-helices in isolation and in proteins.     

Is the hydrophobic core a universal structural element in proteins?

The hydrophobic core, when subjected to analysis based on the fuzzy oil drop model, appears to be a universal structural component of proteins irrespective of their secondary, supersecondary, and tertiary conformations. A study has been performed on a set of nonhomologous proteins representing a variety of CATH categories. The presence of a well-ordered hydrophobic core has been confirmed in each case, regardless of the protein’s biological function, chain length or source organism. In light of fuzzy oil drop (FOD) analysis, various supersecondary forms seem to share a common structural factor in the form of a hydrophobic core, emerging either as part of the whole protein or a specific domain. The variable status of individual folds with respect to the FOD model reflects their propensity for conformational changes, frequently associated with biological function. Such flexibility is expressed as variable stability of the hydrophobic core, along with specific encoding of potential conformational changes which depend on the properties of helices and β-folds.     

Role of A-chain in functioning of the active site of human α-thrombin

This review summarizes current data suggesting that A-chain of the human alpha-thrombin molecule plays a role of allosteric effector in catalytic reactions with various substrates. Special attention is paid to the relationship between A-chain structure and catalytic activity of thrombin. The existence of this relationship is based on studies of natural mutation of A-chain of the alpha-thrombin molecule. Use of molecular and essential dynamics confirmed the role of A-chain in changes of conformation and catalytic properties of this enzyme; these changes involve residues located in the specificity sites and some inserting loops. Current knowledge on structure and properties of thrombin can be used for the development of new antithrombin agents.     

Statistical mechanical treatment of α-helices and extended structures in proteins with inclusion of short- and medium-range interactions

A statistical mechanical model of protein conformation with medium-range interactions between theith and (i+k)^th residues (k<-4) is presented. Two two-state models, an α-helix-coil and an extended-structure-coil model, are formulated using the same form of the partition function, but the two models are applied independently to predict the locations of α-helical, extended, and coil segments; in the relatively few cases (<2%) where the predictions from the two models are in conflict, the prediction is scored as an incorrect one. Two independent sets of statistical weights (one set for each model) are derived to describe the interactions between the 20 amino acid residues for each range of interactionk; they are evaluated by minimizing an objective function so that the probability profiles for the α-helix or extended structure, respectively, in proteins computed from these statistical weights correlate optimally with the experimentally observed native conformations of these proteins. Examination of the resulting statistical weights shows that those for the interactions between hydrophobic residues and between a hydrophobic and a hydrophilic residue have reasonable magnitudes compared to what would be expected from the spatial arrangements of the side chains in the α-helix and the extended structure, and that those for the α-helix-coil model correlate well with experimentally determined values of the Zimm-Bragg parameterss and σ of the helix-coil transition theory. From the point of view of a method to predict the conformational states (i.e., α-helix, extended structure, and coil) of each residue, the statistical weights (as inall empirical prediction schemes) depend very much on the proteins used for the data base, since the presently available set of proteins of known structure is still too small for very high predictability; as a result, the correctness of the prediction is not very good for proteins not included in the data base. However, the correctness of the prediction, at least for the 37 proteins utilized as the data base in this study, is 91% and 87% for the α-helix-coil and the extended-structure-coil models, respectively; further, 79% of all the residues are predicted correctly when both the α-helix-coil and extended-structure-coil models are applied independently.