A minor beta-structured conformation is the active state of a fusion peptide of vesicular stomatitis virus glycoprotein.

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164].     

Molecular dynamics simulations of human prion protein: importance of correct treatment of electrostatic interactions.

Molecular dynamics simulations have been used to investigate the dynamical and structural behavior of a homology model of human prion protein HuPrP(90-230) generated from the NMR structure of the Syrian hamster prion protein ShPrP(90-231) and of ShPrP(<90-231) itself. These PrPs have a large number of charged residues on the protein surface. At the simulation pH 7, HuPrP(90-230) has a net charge of -1 eu from 15 positively and 14 negatively charged residues. Simulations for both PrPs, using the AMBER94 force field in a periodic box model with explicit water molecules, showed high sensitivity to the correct treatment of the electrostatic interactions. Highly unstable behavior of the structured region of the PrPs (127-230) was found using the truncation method, and stable trajectories could be achieved only by including all the long-range electrostatic interactions using the particle mesh Ewald (PME) method. The instability using the truncation method could not be reduced by adding sodium and chloride ions nor by replacing some of the sodium ions with calcium ions. The PME simulations showed, in accordance with NMR experiments with ShPrP and mouse PrP, a flexibly disordered N-terminal part, PrP(90-126), and a structured C-terminal part, PrP(127-230), which includes three alpha-helices and a short antiparallel beta-strand. The simulations showed some tendency for the highly conserved hydrophobic segment PrP(112-131) to adopt an alpha-helical conformation and for helix C to split at residues 212-213, a known disease-associated mutation site (Q212P). Three highly occupied salt bridges could be identified (E146/D144<-->R208, R164<-->D178, and R156<-->E196) which appear to be important for the stability of PrP by linking the stable main structured core (helices B and C) with the more flexible structured part (helix A and strands A and B). Two of these salt bridges involve disease-associated mutations (R208H and D178N). Decreased PrP stability shown by protein unfolding experiments on mutants of these residues and guanidinium chloride or temperature-induced unfolding studies indicating reduced stability at low pH are consistent with stabilization by salt bridges. The fact that electrostatic interactions, in general, and salt bridges, in particular, appear to play an important role in PrP stability has implications for PrP structure and stability at different pHs it may encounter physiologically during normal or abnormal recycling from the pH neutral membrane surface into endosomes or lysomes (acidic pHs) or in NMR experiments (5.2 for ShPrP and 4.5 for mouse PrP).     

NMR structure of a variant human prion protein with two disulfide bridges

The nuclear magnetic resonance structure of the globular domain with residues 121-230 of a variant human prion protein with two disulfide bonds, hPrP(M166C/E221C), shows the same global fold as wild-type hPrP(121-230). It contains three alpha-helices of residues 144-154, 173-194 and 200-228, an anti-parallel beta-sheet of residues 128-131 and 161-164, and the disulfides Cys166-Cys221 and Cys179-Cys214. The engineered extra disulfide bond in the presumed "protein X"-binding site is accommodated with slight, strictly localized conformational changes. High compatibility of hPrP with insertion of a second disulfide bridge in the protein X epitope was further substantiated by model calculations with additional variant structures. The ease with which the hPrP structure can accommodate a variety of locations for a second disulfide bond within the presumed protein X-binding epitope suggests a functional role for the extensive perturbation by a natural second disulfide bond of the corresponding region in the human doppel protein.     

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.     

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

Influence of tertiary structure domain properties on the functionality of apolipoprotein A-I

The tertiary structure of apolipoprotein (apo) A-I and the contributions of structural domains to the properties of the protein molecule are not well defined. We used a series of engineered human and mouse apoA-I molecules in a range of physical-biochemical measurements to address this issue. Circular dichroism measurements of alpha-helix thermal unfolding and fluorescence spectroscopy measurements of 8-anilino-1-napthalenesulfonic acid binding indicate that removal of the C-terminal 54 amino acid residues from human and mouse apoA-I has similar effects; the molecules are only slightly destabilized, and there is a decrease in hydrophobic surface exposure. These results are consistent with both human and mouse apoA-I adopting a two-domain tertiary structure, comprising an N-terminal antiparallel helix bundle domain and a separate less ordered C-terminal domain. Mouse apoA-I is significantly less resistant than human apoA-I to thermal and chemical denaturation; the midpoint of thermal unfolding of mouse apoA-I at 45 degrees C is 15 degrees C lower and the midpoint of guanidine hydrochloride denaturation (D1/2) occurs at 0.5 M as compared to 1.0 M for human apoA-I. These differences reflect the overall greater stability of the helix bundle formed by residues 1-189 in human apoA-I. Measurements of the heats of binding to egg phosphatidylcholine (PC) small unilamellar vesicles and the kinetics of solubilization of dimyristoyl PC multilamellar vesicles indicate that the more stable human helix bundle interacts poorly with lipids as compared to the equivalent mouse N-terminal domain. The C-terminal domain of human apoA-I is much more hydrophobic than that of mouse apoA-I; in the lipid-free state the human C-terminal domain (residues 190-243) is partially alpha-helical and undergoes cooperative unfolding (D1/2 = 0.3 M) whereas the isolated mouse C-terminal domain (residues 187-240) is disordered in dilute solution. The human C-terminal domain binds to lipid surfaces much more avidly than the equivalent mouse domain. Human and mouse apoA-I have very different tertiary structure domain contributions for achieving functionality. It is clear that the stability of the N-terminal helix bundle, and the hydrophobicity and alpha-helix content of the C-terminal domain, are critical factors in determining the overall properties of the apoA-I molecule.     

NMR structure of the human doppel protein

The NMR structure of the recombinant human doppel protein, hDpl(24-152), contains a flexibly disordered "tail" comprising residues 24-51, and a globular domain extending from residues 52 to 149 for which a detailed structure was obtained. The globular domain contains four alpha-helices comprising residues 72-80 (alpha1), 101-115 (alpha2(a)), 117-121 (alpha2(b)), and 127-141 (alpha3), and a short two-stranded anti-parallel beta-sheet comprising residues 58-60 (beta1) and 88-90 (beta2). The fold of the hDpl globular domain thus coincides nearly identically with the structure of the murine Dpl protein. There are close similarities with the human prion protein (hPrP) but, similar to the situation with the corresponding murine proteins, hDpl shows marked local differences when compared to hPrP: the beta-sheet is flipped by 180 degrees with respect to the molecular scaffold formed by the four helices, and the beta1-strand is shifted by two residues toward the C terminus. A large solvent-accessible hydrophobic cleft is formed on the protein surface between beta2 and alpha3, which has no counterpart in hPrP. The helix alpha2 of hPrP is replaced by two shorter helices, alpha2(a) and alpha2(b). The helix alpha3 is shortened by more than two turns when compared with alpha3 of hPrP, which is enforced by the positioning of the second disulfide bond in hDpl. The C-terminal peptide segment 144-149 folds back onto the loop connecting beta2 and alpha2. All but four of the 20 conserved residues in the globular domains of hPrP and hDpl appear to have a structural role in maintaining a PrP-type fold. The conservation of R76, E96, N110 and R134 in hDpl, corresponding to R148, E168, N183 and R208 in hPrP suggests that these amino acid residues might have essential roles in the so far unknown functions of PrP and Dpl in healthy organisms.     

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.     

A role for His155 in binding of human prion peptide144-167 to immobilised prion protein

The interactions and conformational changes that lead to the conversion of the normal prion protein (PrP(c)) to its pathogenic form, PrP(sc), are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP(144-167)) corresponding to residues comprising the alpha helix 1-beta strand 2 domain of PrP(c) is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His(155) in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP(144-167) revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP(144-167).     

Folding intermediates of the prion protein stabilized by hydrostatic pressure and low temperature

Prion diseases are associated with conformational conversion of the cellular prion protein, PrPC, into a misfolded form, PrPSc. We have investigated the equilibrium unfolding of the structured domain of recombinant murine prion protein, comprising residues 121-231 (mPrP-(121-231)). The equilibrium unfolding of mPrP-(121-231) by urea monitored by intrinsic fluorescence and circular dichroism (CD) spectroscopies indicated a two-state transition, without detectable folding intermediates. The fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid (bis-ANS) binds to native mPrP-(121-231), indicating exposure of hydrophobic domains on the protein surface. Increasing concentrations of urea (up to 4 M) caused the release of bound bis-ANS, whereas changes in intrinsic fluorescence and CD of mPrP took place only above 4 M urea. This indicates the existence of a partially unfolded conformation of mPrP, characterized by loss of bis-ANS binding and preservation of the overall structure of the protein, stabilized at low concentrations of urea. Hydrostatic pressure and low temperatures were also used to stabilize partially folded intermediates that are not detectable in the presence of chemical denaturants. Compression of mPrP to 3.5 kbar at 25 degrees C and pH 7 caused a slight decrease in intrinsic fluorescence emission and an 8-fold increase in bis-ANS fluorescence. Lowering the temperature to -9 degrees C under pressure reversed the decrease in intrinsic fluorescence and caused a marked (approximately 40-fold) increase in bis-ANS fluorescence. The increase in bis-ANS fluorescence at low temperatures was similar to that observed for mPrP at 1 atm at pH 4. These results suggest that pressure-assisted cold denaturation of mPrP stabilizes a partially folded intermediate that is qualitatively similar to the state obtained at acidic pH. Compression of mPrP in the presence of a subdenaturing concentration of urea stabilized another partially folded intermediate, and cold denaturation under these conditions led to complete unfolding of the protein. Possible implications of the existence of such partially folded intermediates in the folding of the prion protein and in the conversion to the PrPSc conformer are discussed.     

Membrane insertion of the pore-forming domain of colicin A. A spectroscopic study

In order to gain some insight into the mechanism of insertion into membranes of the pore-forming domain of colicin A and the structure of its membrane-bound form, circular dichroism (in the near and far ultraviolet), fluorescence and ultraviolet spectroscopy experiments were carried out. Because the structure of the water-soluble form of this fragment has been determined by X-ray crystallography, these spectroscopic methods provided valuable information on the secondary structure and the environment of aromatic residues within the two forms of the peptide. These results strongly suggest that the pore-forming domain of colicin A does not undergo drastic unfolding upon insertion into membrane. The conformational change associated with this process is triggered by the negatively charged lipids and probably consists of a reorientation of helix pairs with respect to each other. Exposure of the aromatic residues to the aqueous phase decreases on binding to lipids whilst the exposure of the tryptophans to the membrane phase increases. This cannot occur without a reorientation of helices 3-10. All data from this study support the model presented previously in which the known crystal structure opens like an 'umbrella' inserting the hydrophobic hairpin (helix 8-9) perpendicular to the membrane plane and the helical pair 1-2 and the domain containing the three tryptophans (helices 3-7) lying more or less parallel to the membrane plane. Lipids are bound more tightly to the protein at acidic pH than at neutral pH although a similar lipid protein complex is formed with 1,2-dimyristoyl-sn-glycero(3)-phospho(1)- -sn-glycerol at both pH values.     

Influence of N-terminal helix bundle stability on the lipid-binding properties of human apolipoprotein A-I

As the principal component of high-density lipoprotein (HDL), apolipoprotein (apo) A-I plays essential roles in lipid transport and metabolism. Because of its intrinsic conformational plasticity and flexibility, the molecular details of the tertiary structure of lipid-free apoA-I have not been fully elucidated. Previously, we demonstrated that the stability of the N-terminal helix bundle structure is modulated by proline substitution at the most hydrophobic region (residues around Y18) in the N-terminal domain. Here we examine the effect of proline substitution at S55 located in another relatively hydrophobic region compared to most of the helix bundle domain to elucidate the influences on the helix bundle structure and lipid interaction. Fluorescence measurements revealed that the S55P mutation had a modest effect on the stability of the bundle structure, indicating that residues around S55 are not pivotally involved in the helix bundle formation, in contrast to the insertion of proline at position 18. Although truncation of the C-terminal domain (Δ190-243) diminishes the lipid binding of apoA-I molecule, the mutation S55P in addition to the C-terminal truncation (S55P/Δ190-243) restored the lipid binding, suggesting that the S55P mutation causes a partial unfolding of the helix bundle to facilitate lipid binding. Furthermore, additional proline substitution at Y18 (Y18P/S55P/Δ190-243), which leads to a drastic unfolding of the helix bundle structure, yielded a greater lipid binding ability. Thus, proline substitutions in the N-terminal domain of apoA-I that destabilized the helix bundle promoted lipid solubilization. These results suggest that not only the hydrophobic C-terminal helical domain but also the stability of the N-terminal helix bundle in apoA-I are important modulators of the spontaneous solubilization of membrane lipids by apoA-I, a process that leads to the generation of nascent HDL particles.     

Overexpression and structural study of the cathelicidin motif of the protegrin-3 precursor.

Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence of 96-101 residues, referred to as the cathelicidin motif. The structure of this widespread motif has not yet been reported. The cathelicidin motif of protegrin-3 (ProS) was overexpressed in Escherichia coli as a His-tagged protein to facilitate its purification. The His tag was then removed by thrombin cleavage. In addition, the complete proprotegrin-3 (ProS-PG-3) (120 residues) was overexpressed in baculovirus-infected insect cells. As it contained the antibacterial peptide protegrin-3 in its C-terminal part, ProS-PG-3 contained four disulfide bonds. At neutral pH, ProS and ProS-PG-3 adopted two slowly exchanging conformations that existed in a ratio of 55/45. This ratio was progressively modified at acidic pH to reach a 90/10 value at pH 3.0, suggesting that electrostatic interactions are involved in such a conformational change. Therefore, the structural study of the main conformer was undertaken at pH 3.0 by circular dichroism, mass spectrometry, and homo- and heteronuclear NMR. In parallel, a model for the ProS structure was built from the X-ray structure of the chicken cystatin. ProS and the chicken cystatin share two conserved disulfide bonds as well as a high conservation of hydrophobic residues. The ProS model features the conservation of a hydrophobic core made of the interface between the N-terminal helix and the wrapping beta-sheet. Although the full assignment of the main conformer of ProS could not be obtained, available NMR data validated the presence of the N-terminal helix and of a four-stranded beta-sheet, in agreement with the cystatin fold. Moreover, we clearly demonstrated that ProS and ProS-PG-3 share the same global structure, suggesting that the presence of the highly constrained beta-hairpin of protegrin does not significantly modify the structure of the cathelicidin motif of the protegrin precursor.     

Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase

DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1-171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1-171 of the E. coli DnaB helicase with significant affinity.     

The zinc-dependent redox switch domain of the chaperone Hsp33 has a novel fold

The Escherichia coli chaperone Hsp33 contains a C-terminal zinc-binding domain that modulates activity by a so-called "redox switch". The oxidized form in the absence of zinc is active, while the reduced form in the presence of zinc is inactive. X-ray crystal structures of Hsp33 invariably omit details of the C-terminal domain, which is truncated in protein constructs that are capable of forming crystals. We report the solution structure of a recombinant 61-residue protein containing the zinc-binding domain (residues 227-287) of Hsp33, in the presence of stoichiometric amounts of Zn2+. The zinc-bound protein is well folded, and forms a novel structure unlike other published zinc-binding domains. The structure consists of two helices at right-angles to each other, a two-stranded B-hairpin and a third helix at the C terminus. The zinc site comprises the side-chains of the conserved cysteine residues 232, 234, 262 and 265, and connects a short sequence before the first helix with the tight turn in the middle of the B-hairpin. The structure of the C-terminal zinc-binding domain suggests a mechanism for the operation of the redox switch: loss of the bound zinc ion disrupts the folded structure, allowing the ligand cysteine residues to be oxidized, probably to disulfide bonds. The observation that the C-terminal domain is poorly structured in the active oxidized form suggests that the loss of zinc and unfolding of the domain precedes the oxidation of the thiolate groups of the cysteine residues, since the formation of disulfides between distant parts of the domain sequence would presumably promote the formation of stable three-dimensional structure in the oxidized form.Hsp33 provides an example of a redox signaling system that utilizes protein folding and unfolding together with chemical modification for transduction of external stimuli, in this case oxidative stress, to activate the machinery of the cell that is designed to deal with that stress.     

Assessing the acid-base and conformational properties of histidine residues in human prion protein (125-228) by means of pK(a) calculations and molecular dynamics simulations

A thorough study of the acid-base behavior of the four histidines and the other titratable residues of the structured domain of human prion protein (125-228) is presented. By using multi-tautomer electrostatic calculations, average titration curves have been built for all titratable residues, using the whole bundles of NMR structures determined at pH 4.5 and 7.0. According to our results, (1) only histidine residues are likely to be involved in the first steps of the pH-driven conformational transition of prion protein; (2) the pK(a)'s of His140 and His177 are approximately 7.0, whereas those of His155 and His187 are < 5.5. 10-ns long molecular dynamics simulations have been performed on five different models, corresponding to the most significant combinations of histidine protonation states. A critical comparison between the available NMR structures and our computational results (1) confirms that His155 and His187 are the residues whose protonation is involved in the conformational rearrangement of huPrP in mildly acidic condition, and (2) shows how their protonation leads to the destructuration of the C-terminal part of HB and to the loss of the last turn of HA that represent the crucial microscopic steps of the rearrangement.     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

Unusual property of prion protein unfolding in neutral salt solution

The unfolding of cellular prion protein and its refolding to the scrapie isoform are related to prion diseases. Studies in the literature have shown that structures of proteins, either acidic or basic, are stabilized against denaturation by certain neutral salts, for example, sulfate and fluoride. Contrary to these observations, the full-length recombinant prion protein (amino acid residues 23-231) is denatured by these protein structure stabilizing salts. Under identical concentrations of salts, the structure of the sheep prion protein, which contains a greater number of glycine groups in the N-terminal unstructured segment than the mouse protein, becomes more destabilized. In contrast to the full-length protein, the C-terminal 121-231 prion protein fragment, consisting of all the structural elements of the protein, viz., three alpha-helices and two short beta-strands, is stabilized against denaturation by these salts. We suggest that an increase in the concentration of the anions on the surface of the prion protein molecule due to their preferential interaction with the glycine residues in the N-terminal segment destabilizes the structure of the prion protein by perturbing the prion helix 1 which is the most soluble of all the protein alpha-helices reported so far in the literature. The present results could be relevant to explain the observed structural conversion of the prion protein by anionic nucleic acids and sulfated glycosaminoglycans.     

Native-state conformational dynamics of GART: a regulatory pH-dependent coil-helix transition examined by electrostatic calculations

Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.     

Recombinant collagen studies link the severe conformational changes induced by osteogenesis imperfecta mutations to the disruption of a set of interchain salt bridges

The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the alpha1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly --> Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen.