Increase In Helical Structure Research Articles

Crystal structure of a phosphoribosyl anthranilate isomerase from the hyperthermophilic archaeon Thermococcus kodakaraensis.

A phosphoribosyl anthranilate isomerase, TkTrpF, from Thermococcus kodakaraensis was expressed in Escherichia coli and purified to homogeneity. TkTrpF was crystallized and its structure was determined by molecular replacement in two different space groups (C2 and P1) using data to 1.85 and 1.75 Å resolution, respectively. TkTrpF belongs to the class of TIM-barrel proteins. Structural comparison with other phosphoribosyl anthranilate isomerases (TrpFs) showed the highest structural similarity to Pyrococcus furiosus TrpF. Similarly to P. furiosus TrpF, TkTrpF is a monomer in solution, in contrast to other thermophilic enzymes, which exist as functional dimers. Although in space group P1 TkTrpF crystallizes with two molecules in the asymmetric unit, the interface is highly improbable in solution. Potential factors for the thermostability of TkTrpF were attributed to an increase in helical structure, an increased number of charged residues and an increase in the number of salt bridges.

Construction and Characterization of Vitreoscilla Hemoglobin (VHb) with Enhanced Peroxidase Activity for Efficient Degradation of Textile Dye.

Pollution resulting from the discharge of textile dyes into water systems has become a major global concern. Because peroxidases are known for their ability to decolorize and detoxify textile dyes, the peroxidase activity of Vitreoscilla hemoglobin (VHb) has recently been studied. It is found that VHb and variants of this enzyme show great promise for enzymatic decolorization of dyes and may play a role in achieving their successful removal from industrial wastewater. The level of VHb peroxidase activity correlates with two amino acid residues present within the conserved distal pocket, at positions 53 and 54. In this work, sitedirected mutagenesis of these residues was performed and resulted in improved VHb peroxidase activity. The double mutant, Q53H/P54C, shows the highest dye decolorization and removal efficiency, with 70% removal efficiency within 5 min. UV spectral studies of Q53H/P54C reveals a more compact structure and an altered porphyrin environment (λSoret = 413 nm) relative to that of wild-type VHb (λSoret = 406), and differential scanning calorimetry data indicate that the VHb variant protein structure is more stable. In addition, circular dichroism spectroscopic studies indicate that this variant's increased protein structural stability is due to an increase in helical structure, as deduced from the melting temperature, which is higher than 90°C. Therefore, the VHb variant Q53H/P54C shows promise as an excellent peroxidase, with excellent dye decolorization activity and a more stable structure than wild-type VHb under high-temperature conditions.

The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t1/2) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.

Design, expression, and purification of de novo transmembrane “hairpin” peptides

While our understanding of the folding and structure of water-soluble proteins has progressed to the point where they can be artificially designed and produced from first principles, there has been only limited work toward the de novo design of membrane proteins. Such studies have been hindered in large part due to the practical challenges in the production and characterization of multispanning transmembrane (TM) proteins that arise from their highly hydrophobic character. In this work, we used molecular biology cloning techniques to produce a library of partially randomized Ala- and Ile-rich de novo helix-loop-helix (hairpin) TM constructs as models for tertiary TM-TM folding. From this plasmid DNA library, we selected sequences corresponding to hairpins with 0, 1, or 2 putative TM segments. While purification protocols could be adapted for application with a broad range of designed protein hairpins, bacterial expression of constructs with multiple predicted TM segments was limited as it is with native membrane proteins. Examples of the peptide hairpins obtained were characterized by circular dichroism spectroscopy, tryptophan fluorescence, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We found that hairpins composed of two TM segments display characteristic behavior on detergent solubilization, such as an increase in helical structure (vs. that in aqueous buffer), and sequence-dependent migration rates in SDS-PAGE analysis-features that may serve as structural hallmarks to verify dual TM topology in hairpin sequences.

Syncytial Phenotype of C-Terminally Truncated Herpes Simplex Virus Type 1 gB Is Associated with Diminished Membrane Interactions

The cytoplasmic domain of glycoprotein B (gB) from herpes simplex virus type 1 (HSV-1) is an important regulator of membrane fusion. C-terminal truncations of the cytoplasmic domain lead to either hyperfusion or fusion-null phenotypes. Currently, neither the structure of the cytoplasmic domain nor its mechanism of fusion regulation is known. Here we show, for the first time, that the full-length cytoplasmic domain of HSV-1 gB associates stably with lipid membranes, preferentially binding to membranes containing anionic head groups. This interaction involves a large increase in helical content. However, the truncated cytoplasmic domains associated with the hyperfusion phenotype show a small increase in helical structure and a diminished association with lipid membranes, whereas the one associated with the fusion-null phenotype shows no increase in helical structure and only a minimal association with lipid membranes. We hypothesize that stable binding to lipid membranes is an important part of the mechanism by which the cytoplasmic domain negatively regulates membrane fusion. Moreover, our experiments with truncated cytoplasmic domains point to two specific regions that are critical for membrane interactions. Taken together, our work provides several important new insights into the architecture of the cytoplasmic domain of HSV-1 gB and its interaction with lipid membranes.

Mimicking the Plant Cell Interior under Water Stress by Macromolecular Crowding: Disordered Dehydrin Proteins Are Highly Resistant to Structural Collapse

The dehydrins are a class of drought-induced proteins in plants that lack a fixed three-dimensional structure. Their specific molecular action, as well as the reason for their disordered character, is as yet poorly understood. It has been speculated, however, that the dehydrins are tuned to acquire a biologically active structure only under the conditions in which they normally function (i.e. upon dehydration). To test this hypothesis, we here investigate the effect of reduced water content and macromolecular crowding on three dehydrins from Arabidopsis (Arabidopsis thaliana). As a simplistic model for mimicking cellular dehydration, we used polyethylene glycol, glycerol, and sugars that plants naturally employ as compatible solutes (i.e. sucrose and glucose). Macromolecular crowding was induced by the large polysaccharides Ficoll and dextran. The results show that the dehydrins are remarkably stable in their disordered state and are only modestly affected by the solvent alterations. A notable exception is the dehydrin Cor47, which shows a small, intrinsic increase in helical structure at high concentrations of osmolytes. We also examined the effect of phosphorylation but found no evidence that such posttranslational modifications of the dehydrin sequences modulate their structural response to osmolytes and crowding agents. These results suggest that the dehydrins are highly specialized proteins that have evolved to maintain their disordered character under conditions in which unfolded states of several globular proteins would tend to collapse.

TPR-Mediated Self-Association of Plant SGT1

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants. We show that recombinant Sgt1 from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgt1 does not. Further experiments on barley Sgt1 demonstrate unambiguously a TPR-mediated dimerization, which is concentration- and ionic-strength-dependent and results in a global increase in helical structure and stability of the protein. Dimerization is also redox sensitive, being completely abolished by the formation of an intramolecular disulfide bond where the contributing cysteines are conserved in plant Sgt1s. The dimer interface was mapped through cross-linking and mass spectrometry to the C-terminal region of the TPR domain. Our study, which provides the first biophysical characterization of plant Sgt1, highlights how TPR domains can mediate self-association in solution and that sequence variation in the regions involved in oligomerization affects the propensity of TPR-containing proteins to dimerize.

The Intermediate Chain of Cytoplasmic Dynein Is Partially Disordered and Gains Structure upon Binding to Light-Chain LC8

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.

Aib-based peptide backbone as scaffolds for helical peptide mimics.

Helical peptides that can intervene and disrupt therapeutically important protein-protein interactions are attractive drug targets. In order to develop a general strategy for developing such helical peptide mimics, we have studied the effect of incorporating alpha-amino isobutyric acid (Aib), an amino acid with strong preference for helical backbone, as the sole helix promoter in designed peptides. Specifically, we focus on the hdm2-p53 interaction, which is central to development of many types of cancer. The peptide corresponding to the hdm2 interacting part of p53, helical in bound state but devoid of structure in solution, served as the starting point for peptide design that involved replacement of noninteracting residues by Aib. Incorporation of Aib, while preserving the interacting residues, led to significant increase in helical structure, particularly at the C-terminal region as judged by nuclear magnetic resonance and circular dichroism. The interaction with hdm2 was also found to be enhanced. Most interestingly, trypsin cleavage was found to be retarded by several orders of magnitude. We conclude that incorporation of Aib is a feasible strategy to create peptide helical mimics with enhanced receptor binding and lower protease cleavage rate.

High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners.

High-risk papillomaviruses are known to exert their transforming activity mainly through E7, one of their two oncoproteins. Despite its relevance, no structural information has been obtained that could explain the apparent broad binding specificity of E7. Recombinant E7 from HPV-16 purified to near homogeneity showed two species in gel filtration chromatography, one of these corresponding to a dimer with a molecular weight of 22 kDa, determined by multiangle light scattering. The E7 dimer was isolated for characterization and was shown to undergo a substantial conformational transition when changing from pH 7.0 to 5.0, with an increase in helical structure and increased solvent accessibility to hydrophobic surfaces. The protein was resistant to thermal denaturation even in the presence of SDS, and we show that persistent residual structure in the monomer is responsible for its reported anomalous electrophoretic behavior. The dimer also displays a nonglobular hydrodynamic volume based on gel filtration experiments and becomes more globular in the presence of 0.3 M guanidinium chloride, with hydrophobic surfaces becoming accessible to the solvent, as indicated by the large increase in ANS binding. At low protein concentration, dissociation of the globular E7 dimer was observed, preceding the cooperative unfolding of the structured and extended monomer. Although E7 bears properties that resemble natively unfolded polypeptides, its far-UV circular dichroism spectrum, cooperative unfolding, and exposure of ANS binding sites support a folded and extended, as opposed to disordered and fluctuating, conformation. The large increase in solvent accessibility to hydrophobic surfaces upon small pH decrease within physiological range and in mild denaturant concentrations suggests conformational properties that could have evolved to enable protein-protein recognition of the large number of cellular binding partners reported.

Initial studies of the equilibrium folding pathway of staphylococcal nuclease

Spectroscopic methods were used to examine the sequential build up of structure in the denatured state of staphylococcal nuclease. The 'free energy distance' between the native and denatured states was manipulated by altering conditions in solution (for example altering urea or glycerol concentration) and by changing the amino acid sequences. Initial studies employed a fragment of nuclease, referred to as delta 131 delta, which lacks six structural residues from the amino terminus and one structural residue from the carboxy-terminus. Nuclear magnetic resonance analysis of this fragment in solution revealed a modest quantity of dynamic structure which is native-like in character. With the addition of urea, 12 new HN peaks appeared in the 1H-15N correlation spectrum, presumably as a result of the breakdown of residual structure involving the first three beta strands. With the addition of glycerol, there was a rapid increase in the quantity of beta sheet structure detected by circular dichroism spectroscopy. At very high glycerol concentrations, an increase in helical structure became apparent. These data in addition to previously published results suggest that: (i) a beta-meander (strands beta 1-beta 2-beta 3) and the second alpha helix (alpha 2) are among the most stable local structures; (ii) the five-strand beta-barrel forms in a reaction which does not require the presence of several other native substructures; and (iii) the last step on the equilibrium folding pathway may be the formation and packing of the carboxy terminal alpha helix (alpha 3) to give the native state.

Konformationsstudien an Partialsequenzen von ACTH und Analogen. Theoretische Vorhersagen und CD-Untersuchungen an Polyethylenglykol-gebundenen Oligopeptiden / Conformational Studies on Partial Sequences of ACTH and Analogues. Theoretical Predictions and CD Investigations of Polyethylene glycol)-bound Oligopeptides

Abstract Conformational studies on synthetic segments)* of ACTH using circular dichroism (CD) are described. The segments [Val4]-ACTH (1-10) = I, [Pro1, Ala2, Ala3, Val4]-ACTH (1-10) = II and ACTH (11-23) = III have been synthesized according to the Liquid-Phase-Method with polyethylene glycol (PEG) as solubilizing C-terminal protecting group. The peptide PEG esters were studied in water and 2,2,2-trifluoroethanol (TFE) and the dependence of chain length, side chain protection and solvent upon the conformation has been evaluated. From these measurements, I shows partially helical structure in TFE and β-conformation in H2O. The modified 10-peptide II with strong helix formers at the N-terminus shows substantial amounts of helical structure in TFE and in water. A significant increase in helical structure is observed by adding the Pro residue to the N-terminus. The CD spectra of the 13-peptide III ist also typical for partially helical structure, however other types of ordered conformations cannot be excluded. The experimental data are compared with the results from calculations of the secondary structure deduced from the empirical prediction scheme of Chou and Fasman and the more elaborated statistical mechanical treatment of peptide conformations proposed by Tanaka and Scheraga. The agreement between experimental data and theoretical prediction is reason-able for I and II, whereas III is expected to adopt extended conformations beside unordered forms. The experimentally found unique behaviour of Prolin was rationalized by the asymmetric helix nucleation feature of this residue. A model for the most probable conformation of ACTH is proposed and a critical evaluation of the predictive schemes is given.

Optical rotatory dispersion and spectrophotometric studies on fetuin in solutions containing detergent and denaturing reagents.

Abstract The effects of the denaturing agents, urea and guanidine·HCl, and detergents, sodium dodecyl sulphate and hexadecyltrimethylammonium bromide (HTAB), on the optical rotatory dispersion of fetuin have been investigated. It was concluded that fetuin has a high affinity for the cationic detergent HTAB which seems to bind, at one type of binding site only, as demonstrated by both an optical rotatory and Optical density study. The same detergent also increased by the parameters λc and b o , determined from Yang-Doty plots and the Moffitt equation, respectively. The conformation-dependent Cotton trough also shows an increase in amplitude and in addition undergoes a shift to higher wavelengths upon addition of HTAB. Although these observations suggest an increase in helical structure, caution was exercised in interpreting the results in this way since optical artifacts (e.g., higher refractive indexforprotein-micelle complex) may contribute. Modification of the fetuin molecule by removal of sialic acid or cleavage of the disulfide bonds results in a reduction in helical content of the molecule but does not alter the affinity of the protein for HTAB. The effect of HTAB on the various fetuin preparations was opposed by guanidine·HCl but not by urea; this was attributed to a charge effect by the former ions. The red shifts of the Cotton curve and the absorption spectra, upon the addition of HTAB to fetuin solutions are gradual and seem to be related, and it is tempting to attribute both phenomena to possible micelle formation in which tyrosine chromophores are also included. Such protein-micellar complexes would have a higher refractive index than the bulk solvent, and this in itself would cause a red shift of the π → π * absorption band of the helix. On the basis of solvent perturbation and spectrophotometric studies, it was concluded that fetuin contains no buried tyrosine groups. A structural similarity of both bovine albumin and fetuin was proposed as a result of the appearance in both cases of a second Cotton trough in the far-ultraviolet region after cleaving the disulfide bonds, indicative in both cases of a more random conformation.