Decrease In Solvent Accessibility Research Articles

Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.

Random Coils of Proteins Situated Between a Beta Strand and an Alpha Helix Demonstrate Decreased Solvent Accessibility.

Surface accessibility of different types of the same elements of secondary structure has been studied in 10 non-redundant sets of proteins (total number of three-dimensional structures is 1730) with a help of DSSP (Dictionary of Secondary Structure of Proteins). Random coils (C), beta strands (B), and alpha helices (H) have been classified according to their flanking elements of secondary structure in a polypeptide chain. Thanks to this kind of classification, for the first time it has been shown that random coils situated between a beta strand and an alpha helix (BCH) contain significantly lower fraction of exposed residues compared to other types of random coils; the least accessible beta strands are situated between two alpha helices (HBH), and the least accessible alpha helices are situated between a beta strand and an alpha helix (BHH). Discovered trends are explained as consequences of the natural selection that had been stabilizing the secondary structure of proteins on early steps of their evolution. Acquired differences in amino acid content of different types of random coils, alpha helices, and beta strands led to the formation of partially buried but hydrophilic BCH random coils because of their enrichment by Ser, Thr, and Asp residues. As a result, BCH random coils became prone to bind cations because of their lower hydration and decreased usage of positively charged amino acid residues. The mechanism described above led to the formation of active centers in ancient metalloenzymes. Nowadays one can observe decreased surface accessibility of amino acid residues in BCH random coils, in HBH beta strands, and in BHH alpha helices in proteins possessing hydrophobic cores.

Measuring the Energy Barrier of the Structural Change That Initiates Amyloid Formation

Obtaining kinetic and thermodynamic information for protein amyloid formation can yield new insight into the mechanistic details of this biomedically important process. The kinetics of the structural change that initiates the amyloid pathway, however, has been challenging to access for any amyloid protein system. Here, using the protein β-2-microglobulin (β2m) as a model, we measure the kinetics and energy barrier associated with an initial amyloidogenic structural change. Using covalent labeling and mass spectrometry, we measure the decrease in solvent accessibility of one of β2m's Trp residues, which is buried during the initial structural change, as a way to probe the kinetics of this structural change at different temperatures and under different amyloid forming conditions. Our results provide the first-ever measure of the activation barrier for a structural change that initiates the amyloid formation pathway. The results also yield new mechanistic insight into β2m's amyloidogenic structural change, especially the role of Pro32 isomerization in this reaction.

Expression and Purification of Protease-Activated Receptor 4 (PAR4) and Analysis with Histidine Hydrogen-Deuterium Exchange.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteolysis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine hydrogen-deuterium exchange. PAR4 has nine histidines that are spaced throughout the protein, allowing a global view of solvent accessible and nonaccessible regions. Peptides containing each of the nine His residues were used to determine the t1/2 for each His residue in apo or thrombin-activated PAR4. The thrombin-cleaved PAR4 exhibited a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380), demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin-cleaved PAR4 also was resistant to thermolysin digestion. In contrast, the rate of thermolysin proteolysis following stimulation with the PAR4 activation peptide was the same as that of unstimulated PAR4. Further analysis showed the C-terminus is protected in thrombin-activated PAR4 compared to uncleaved or agonist peptide-treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.

Impact of a non-synonymous Q281R polymorphism on structure of human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2 ).

Non-synonymous single nucleotide polymorphisms (nsSNPs) are genetic variations at single base resulting in an amino acid change which have been associated with various complex human diseases. The human Lipoprotein-associated phospholipase A2 (Lp-PLA2 ) gene harbours a rare Q281R polymorphism which was previously reported to cause loss of enzymatic function. Lp-PLA2 is an important enzyme which catalyzes the hydrolysis of polar phospholipids releasing pro-atherogenic and pro-inflammatory mediators involved in the pathogenesis of atherosclerosis. Our current study is aimed at elucidating the structural and functional consequences of Q281R polymorphism on Lp-PLA2 . The Q281R mutation is classified as deleterious and causes protein instability as deduced from evolutionary, folding free energy changes and Support vector machine (SVM)-based methods. A Q281R mutant structure was deciphered using homology modelling approach and was validated using phi and psi dihedral angles distribution, ERRAT, Verify_3D scores, Protein Structure Analysis (ProSA) energ,y and Z-score. A decreased hydrophobic interactions and weaker substrate binding affinity was observed in the mutant compared to the wild- type (WT) using molecular docking. Further, the mutant displayed enhanced structural flexibility particularly in the low density lipoprotein (LDL) binding domain, decreased solvent accessibility of catalytic residues-Phe274 and Ser273 and increased Cɑ distance between Phe274 and Leu153 and large conformational entropy change as inferred from all-atom molecular dynamics (MD) simulation and essential dynamics (ED) studies. Our results corroborate well with previous experimental studies and thus these aberrations in the Q281R mutant structure may help explain the molecular basis of loss of enzyme activity.

Catalytic modulation of human cytochromes P450 17A1 and P450 11B2 by phospholipid

Unlike most of the drug-metabolizing cytochrome P450s, microsomal P450 17A1 and mitochondrial P450 11B2 catalyze sequential multi-step reactions in steroid biosynthesis. The membrane phospholipid composition might be one parameter that modulates the efficiency and processivity of specific pathways. Here we systematically examined the effects of physiologically relevant phospholipids on the catalysis of purified P450 17A1, P450 11B2, and P450 11B1 in reconstituted assay systems. Both dioleoylphosphatidylcholine (DOPC, 18:1) and dilauroylphosphatidylcholine (DLPC, 12:0) were found to be very efficient in reconstituting 17-hydroxylase and 1720-lyase reactions of P450 17A1. Phosphatidylethanolamine (PE) specifically enhanced 1720-lyase activity up to 2.4-fold in the presence of phosphatidylcholine. On the other hand, P450 11B2-catalyzed production of aldosterone from 11-deoxycorticosterone was very low and from 18-hydroxycorticosterone nil, implying low processivity. DOPC or cardiolipin, which is exclusively located in the inner mitochondrial membrane, maximized aldosterone yield. In sharp contrast, reconstitution of homologous P450 11B1 with DOPC significantly decreased corticosterone formation without affecting the synthesis of 18-hydroxycorticosterone. The intrinsic fluorescence of P450 17A1 and 11B2 increased in the presence of DOPC, DLPC and PE. Acrylamide quenching studies showed that PE decreased solvent accessibility for tryptophan in P450 17A1, as did 20:4 PC or 18:2 PC for P450 11B2. A moderately positive correlation between the proportion of high-spin substrate-bound species and catalytic activity was only observed in the presence of phosphatidylcholines with low-temperature phase transition. These results demonstrate the potential for phospholipids to regulate the activity of steroidogenic P450 activities and thereby steroid hormone biosynthetic pathways.

In Search of Functional Advantages of Knots in Proteins.

We analysed the structure of deeply knotted proteins representing three unrelated families of knotted proteins. We looked at the correlation between positions of knotted cores in these proteins and such local structural characteristics as the number of intra-chain contacts, structural stability and solvent accessibility. We observed that the knotted cores and especially their borders showed strong enrichment in the number of contacts. These regions showed also increased thermal stability, whereas their solvent accessibility was decreased. Interestingly, the active sites within these knotted proteins preferentially located in the regions with increased number of contacts that also have increased thermal stability and decreased solvent accessibility. Our results suggest that knotting of polypeptide chains provides a favourable environment for the active sites observed in knotted proteins. Some knotted proteins have homologues without a knot. Interestingly, these unknotted homologues form local entanglements that retain structural characteristics of the knotted cores.

Analysis of Protease Activated Receptor 4 (PAR4) By Histidine Hydrogen Deuterium Exchange

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have central roles in the cardiovascular system. Numerous studies have focused on the signaling pathways mediated by PARs; however, the structural rearrangements that initiate these pathways are unknown. PARs are activated when proteolytic cleavage exposes a tethered ligand that interacts with a binding pocket on the receptor. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. We now describe the first biophysical studies examining this unique mode of activation using PAR4. PAR4 is one of the thrombin receptors on platelets and is required for sustained platelet activation. Recent data from our laboratory and others suggest that PAR4 has the potential to be a therapeutic target. To examine the tethered ligand activation mechanism in general and further examine PAR4 as a target, we have expressed and purified PAR4 that does not contain additional stabilizing sequences from Sf9 cells for biophysical studies. The current study uses histidine-hydrogen deuterium exchange (His-HDX). His-HDX is a structural mass spectrometry technique that relies on the slow exchange of the C2 carbon on the imidazole ring of histidine. PAR4 has nine histidine residues that are well spaced throughout the protein, which gives us a global view of solvent accessible and non-accessible regions. Unique peptides were generated that contained each of the histidine residues (His136, His159, His180, His229, His240, His269, His280, His306, and His380). These peptides were used to determine the t1/2 for each His residue in full length or thrombin cleaved PAR4. The t1/2 values for His159 and His269 were greater than 40 hours in both states indicating that these two histidine residues are not exposed to solvent in either case. In contrast, the thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have a decrease in solvent accessibility upon thrombin treatment. These residues are spread throughout the protein, which indicates that PAR4 undergoes a global conformational change and the overall structure becomes more rigid. In agreement with these data, thrombin cleaved PAR4 is more resistant to protease digestion by chymotrypsin. The half-life was extended 4-fold over the uncleaved PAR4. We are also able to make conclusions regarding specific regions of PAR4. His229 is located in extracellular loop 2 near the proposed ligand-binding site and has a 2-fold increase in t1/2 following thrombin treatment. These data suggest that the tethered ligand may be binding to this region and protecting it from exchange. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysical approaches. These studies shed light on the overall conformational changes that follow activation of PARs by a protease. Finally, the His-HDX used for purified PAR4 lays important groundwork that will allow us to examine PAR4 and other platelet GPCRs in their native membrane environment using this and related mass spectrometry approaches. DisclosuresNo relevant conflicts of interest to declare.

Interaction of Mycobacterium tuberculosis RshA and SigH is mediated by salt bridges.

The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1–25, 58–69, 90–111, 115–132 and 157–196 and residues 35–57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.

Size-Dependent Interactions between Au Nanoparticles and DNA in Electrochemical Oxidation by Metal Complexes

Single-stranded oligonucleotides (ssDNA) containing guanine bases in their sequences were adsorbed onto gold nanoparticles (AuNPs) by electrostatic interaction. Cyclic voltammetry of Ru(bpy)32+ in the presence of the ssDNA-AuNP complex resulted in an enhanced anodic current due to the oxidation of the guanine bases of DNA. The current obtained with ssDNA-AuNP appeared much smaller than the corresponding ssDNA alone. This current reduction was due to the decrease in solvent accessibility of the guanines in ssDNA immobilized to AuNPs. A progressive decrease in the current was observed in the titration of AuNPs to ssDNA, and a minimum current was eventually obtained, indicating complete binding of ssDNA. The size dependences of AuNPs on the interaction between ssDNA and AuNP were also studied, and the ssDNA adsorbed to 5 nm AuNPs was more solvent-accessible for the Ru mediator than 13 and 30 nm AuNPs.

Conformational Changes in Myosin V Monitored by Hydrogen-Deuterium Exchange Mass Spectrometry

Conformational changes between the apo and the BeF.ADP states of Myosin V were determined by hydrogen/deuterium exchange monitored by FT-ICR mass spectrometry.In the BeF.ADP state we have observed opening of the 50K domain cleft (actin binding cleft) (aa 566-578) as expected from the x-ray structure (Coureux et al., Nature 2003). The opening of the actin binding cleft was coupled to the closure (decreased solvent accessibility) of the active site - a stretch of aa 197-231 that includes switch 1, loop1 and B6 β-strand and an additional peptide at the entrance to the active site (aa 233-237).Unexpected changes were observed in the in the SH1 helix (aa 678-692), which was more exposed and the upper 50 kDa domain (aa 331-339 and 350-381) which was less exposed. More protection was observed in the loop coupling B3 β-strand to the SH1 helix (aa 662-676).Figure: Conformational changes in Myosin V mapped on the crystal structure. Increased solvent accessibility in BeF.ADP in blue wide cylinders. Decreased accessibility in narrow red cylinders.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4

The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.

A Mass Spectrometric Approach to the Study of DNA-Binding Proteins: Interaction of Human TRF2 with Telomeric DNA

Human telomeric repeat binding factor 2 (hTRF2) is a protein that plays an important role in capping human telomeres to protect them from DNA damage repair systems. The ineffectiveness of hTRF2 may be linked to aging and cancer. We report the use of PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) and selective acetylation of lysine residues to study the interaction of the DNA-binding domain and double-stranded telomeric DNA (repeats of TTAGGG). By increasing the resolution of PLIMSTEX to the peptide level, we localized the changes in deuterium uptake of hTRF2 as a function of varying amounts of a model oligodeoxynucleotide. From these experiments, we determined the affinity constant for binding to DNA, which is within a factor of 3 of the previously reported value. Amide H/D exchange revealed portions of the protein that have contacts with the phosphate backbone of DNA, whereas acetylation disclosed the decrease in solvent accessibility of regions containing Lys 447 and 488, which must be involved in interactions with the DNA major and minor grooves. These complementary approaches of amide H/D exchange and selective side chain modification can be employed effectively to pinpoint and quantify protein-ligand, in particular protein-DNA, interactions.

Allosteric Mechanisms in Cytochrome P450 3A4 Studied by High-Pressure Spectroscopy: Pivotal Role of Substrate-Induced Changes in the Accessibility and Degree of Hydration of the Heme Pocket

Allosteric mechanisms in human cytochrome P450 3A4 (CYP3A4) in oligomers in solution or monomeric enzyme incorporated into Nanodiscs (CYP3A4ND) were studied by high-pressure spectroscopy. The allosteric substrates 1-pyrenebutanol (1-PB) and testosterone were compared with bromocriptine (BCT), which shows no cooperativity. In both CYP3A4 in solution and CYP3A4ND, we observed a complete pressure-induced high-to-low spin shift at pressures of <3 kbar either in the substrate-free enzyme or in the presence of BCT. In addition, both substrate-free and BCT-bound enzyme revealed a pressure-dependent equilibrium between two states with different barotropic parameters designated R for relaxed and P for pressure-promoted conformations. This pressure-induced conformational transition was also observed in the studies with 1-PB and testosterone. In CYP3A4 oligomers, the transition was accompanied by an important increase in homotropic cooperativity with both substrates. Surprisingly, at high concentrations of allosteric substrates, the amplitude of the spin shift in both CYP3A4 in solution and Nanodiscs was very low, demonstrating that hydrostatic pressure induces neither substrate dissociation nor an increase in the heme pocket hydration in the complexes of the pressure-promoted conformation of CYP3A4 with 1-PB or testosterone. These findings suggest that the mechanisms of interactions of CYP3A4 with 1-PB and testosterone involve an effector-induced transition that displaces a system of conformational equilibria in the enzyme toward the state(s) with decreased solvent accessibility of the active site so that the flux of water into the heme pocket is impeded and the high-spin state of the heme iron is stabilized.

The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.

Structural Dynamics of the Actin–Myosin Interface by Site-directed Spectroscopy

We have used site-directed spin and fluorescence labeling to test molecular models of the actin-myosin interface. Force is generated when the actin-myosin complex undergoes a transition from a disordered weak-binding state to an ordered strong-binding state. Actomyosin interface models, in which residues are classified as contributing to either weak or strong binding, have been derived by fitting individual crystallographic structures of actin and myosin into actomyosin cryo-EM maps. Our goal is to test these models using site-directed spectroscopic probes on actin and myosin. Starting with Cys-lite constructs of both yeast actin (ActC) and the Dictyostelium myosin II motor domain (S1dC), site-directed labeling (SDL) mutants were generated by mutating residues to Cys in the proposed weak and strong-binding interfaces. This report focuses on the effects of forming the strong-binding complex on four SDL mutants, two located in the proposed weak-binding interface (ActC5 and S1dC619) and two located in the proposed strong-binding interface (ActC345 and S1dC401). Neither the mutations nor labeling prevented strong actomyosin binding or actin-activation of myosin ATPase. Formation of the strong-binding complex resulted in decreased spin and fluorescence probe mobility at all sites, but both myosin-bound probes showed remarkably high mobility even after complex formation. Complex formation decreased solvent accessibility for both actin-bound probes, but increased it for the myosin-bound probes. These results are not consistent with a simple model in which there are discrete weak and strong interfaces, with only the strong interface forming under strong-binding conditions, nor are they consistent with a model in which surface residues become rigid and inaccessible upon complex formation. We conclude that all four of these residues are involved in the strong actin-myosin interface, but this interface is remarkably dynamic, especially on the surface of myosin.

Influence of the Dimer Interface on Glutathione Transferase Structure and Dynamics Revealed by Amide H/D Exchange Mass Spectrometry

Mammalian glutathione (GSH) transferases are dimeric proteins, many of which share a common hydrophobic interaction motif that is important for dimer stability. In the rGSTM1-1 enzyme this motif involves the side chain of F56, located on the 56 loop of the N-terminal domain, which is intercalated between the alpha4- and alpha5-helices of the C-terminal domain of the opposing subnuit. Disruption of the complementary interactions in this motif by mutation of F56 to serine, arginine, or glutamate is known to have deleterious effects on catalytic efficiency but remarkably different effects on the stability of the dimer [Hornby et al. (2002) Biochemistry 41, 14238-14247]. The structural basis for the behavior of the mutants by amide H/D exchange mass spectrometry is described. A substantial decrease in H/D exchange is observed in the GSH binding domain and in parts of the dimer interface upon substrate binding. The F56S and F56R mutants exhibit enhanced H/D exchange kinetics in the GSH binding domain and at the dimer interface. In contrast, the F56E mutant shows a decrease in the rate and extent of amide H/D exchange at the dimer interface and enhanced exchange kinetics in the GSH binding domain. The results suggest that the F56E mutant has a restructured dimer interface with decreased solvent accessibility and dynamics. Although all of the F56 mutations disrupt the GSH binding site, the effects of the mutations on the structure of the subunit interface and dimer stability are quite distinct.

Ultraviolet Resonance Raman Studies Reveal the Environment of Tryptophan and Tyrosine Residues in the Native and Partially Folded States of the E Colicin-Binding Immunity Protein Im7

Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.

Crystal Structure of S-ovalbumin as a Non-loop-inserted Thermostabilized Serpin Form

Ovalbumin, a non-inhibitory member of serine proteinase inhibitors (serpin), is transformed into a heat-stabilized form, S-ovalbumin, under elevated pH conditions. The structural mechanism for the S-ovalbumin formation has long been a puzzling question in food science and serpin structural biology. On the basis of the commonly observed serpin thermostabilization by insertion of the reactive center loop into the proximal beta-sheet, the most widely accepted hypothetical model has included partial loop insertion. Here we demonstrate, for the first time, the crystal structure of S-ovalbumin at 1.9-A resolution. This structure unequivocally excludes the partial loop insertion mechanism; the overall structure, including the reactive center loop structure, is almost the same as that of native ovalbumin, except for the significant motion of the preceding loop of strand 1A away from strand 2A. The most striking finding is that Ser-164, Ser-236, and Ser-320 take the d-amino acid residue configuration. These chemical inversions can be directly related to the irreversible and stepwise nature of the transformation from native ovalbumin to S-ovalbumin. As conformational changes of the side chains, significant alternations are found in the values of the chi 1 of Phe-99 and the chi 3 of Met-241. The former conformational change leads to the decreased solvent accessibility of the hydrophobic core around Phe-99, which includes Phe-180 and Phe-378, the highly conserved residues in serpin. This may give a thermodynamic advantage to the structural stability of S-ovalbumin.

Crystal Structures at Atomic Resolution Reveal the Novel Concept of “Electron-harvesting” as a Role for the Small Tetraheme Cytochrome c

The genus Shewanella produces a unique small tetraheme cytochrome c that is implicated in the iron oxide respiration pathway. It is similar in heme content and redox potential to the well known cytochromes c(3) but related in structure to the cytochrome c domain of soluble fumarate reductases from Shewanella sp. We report the crystal structure of the small tetraheme cytochrome c from Shewanella oneidensis MR-1 in two crystal forms and two redox states. The overall fold and heme core are surprisingly different from the soluble fumarate reductase structures. The high resolution obtained for an oxidized orthorhombic crystal (0.97 A) revealed several flexible regions. Comparison of the six monomers in the oxidized monoclinic space group (1.55 A) indicates flexibility in the C-terminal region containing heme IV. The reduced orthorhombic crystal structure (1.02 A) revealed subtle differences in the position of several residues, resulting in decreased solvent accessibility of hemes and the withdrawal of a positive charge from the molecular surface. The packing between monomers indicates that intermolecular electron transfer between any heme pair is possible. This suggests there is no unique site of electron transfer on the surface of the protein and that electron transfer partners may interact with any of the hemes, a process termed "electron-harvesting." This optimizes the efficiency of intermolecular electron transfer by maximizing chances of productive collision with redox partners.