Compact form of DNA induced by DNA-binding protein HU.

Interaction of DNA-binding protein HU from Bacillus stearothermophilus (HUBst) with coliphage T2 DNA was investigated by means of a single-duplex DNA chain visualization method using fluorescence microscopy. Fluorescence microscopic images of coliphage T2 DNA molecules were observed as a function of HUBst concentration. The average fluorescence image size of T2 DNA decreased with increase in HUBst concentration to a size comparable to that of a DNA globule induced by polyethylene glycol (PEG) and multivalent cation (MVC). The change to globule-like DNA proceeded gradually and monotonously, in contrast to the coil-globule transition of DNA induced by PEG and MVC. The histogram of the fluorescence image length was essentially a single-modal one throughout the process of conformational change. These results indicate that the process of shrinking of DNA from a random coil to a globule-like one is not of a transitional nature. The interaction of HUBst with DNA and the mechanism of shrinkage are concluded to be different from those of PEG-induced and MVC-induced coil-globule transition of DNA.     

Reactivity of sarcoplasmic reticulum adenosinetriphosphatase with iodoacetamide spin-label: evidence for two conformational states of the substrate binding sites

The labeling kinetics of sarcoplasmic reticulum ATPase with the iodoacetamide spin probe N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide were followed under conditions designed to selectively label all reactive groups. Approximately 1 mol of spin-label reacted per one 100 000-dalton ATPase chain, indicating only one residue on the enzyme had been labeled. One uniform rate of labeling was observed in the presence of Ca2+. When substrate was then added, approximately one-half of the residues showed a 10-fold increase in labeling rate while the remaining residues reacted at the initial, slower rate. Sequential labeling experiments further established that the two labeling rates correspond to the coexistence of two conformational state of the enzyme. Both Ca2+ and substrate are required to obtain an equal distribution between states, and the effect is completely reversed when substrate is removed. The iodoacetamide spin probe is known to be highly sensitive to the conformation of the ATPase binding pocket, and the residue labeled here is the one which generates broadening in the electron paramagnetic resonance spectrum on substrate binding. Due to the unique selectively of the labeling reaction, it is suggested that when both substrate and Ca2+ are bound to the enzyme, conditions which are precursory to enzyme phosphorylation, two specific conformations of the binding pocket exist in approximately at 50:50 ratio.     

Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane

Collagen type IV forms a network in the basement membrane into which other constituents of the tissue are incorporated. It also provides cell-adhesion sites that are specifically recognized by cell-surface receptors, i.e., the integrins. Different from the ubiquitous sequential RGD adhesion motif found in most of the matrix proteins, in collagen type IV, the responsible binding sites for alpha1beta1 integrin have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. Because of the heterotrimeric character of this collagen, the spatial geometry of the binding epitope depends not only on the triple-helical fold, but decisively even on the stagger of the chains. To investigate the effects of chain registration on the conformational properties and binding affinities of this adhesion epitope, two synthetic heterotrimeric collagen peptides consisting of the identical three chains were assembled by an artificial cystine knot in two different registers, i.e., in the most plausible alpha2alpha1alpha1' and less probable alpha1alpha2alpha1' chain alignment. A detailed conformational characterization of both trimers allowed to correlate their different binding affinities for alpha1beta1 integrin with the degree of local plasticity of the two different triple helices. Optimal local breathing of the rod-shaped collagens is apparently crucial for selective recognition by proteins interacting with these main components of the extracellular matrix.     

Differences in dynamic structure of LC8 monomer, dimer, and dimer-peptide complexes

Dimerization of dynein light chain LC8 creates two symmetric grooves at the dimer interface with diverse binding capabilities. In addition to pH and protein concentration, dimerization is affected by phosphorylation, as illustrated by a phosphomimetic mutation that promotes dissociation of LC8 to a monomer and subsequent dissociation from the dynein complex in vitro. In this work we characterize the dynamic structure and unfolding profiles of an LC8 mutant, H55K, as a model for monomeric LC8 at neutral pH. Backbone (15)N relaxation experiments show that the monomer, while primarily ordered, has more heterogeneous dynamics relative to the LC8 dimer, predominantly in residues that ultimately form the binding groove, particularly those in beta 1 and beta 3 strands. This heterogeneity suggests that conformations that are primed for binding are sampled in the inactive monomer and favored in the active dimer. Further changes of LC8 backbone dynamics upon binding to short peptides from Swallow (Swa) and dynein intermediate chain (IC) were elucidated. The conformational heterogeneity apparent in the LC8 dimer is retained in LC8/IC but is lost in LC8/Swa, suggesting that the degree of ordering upon binding is ligand dependent. The reduced complexity of motion in LC8/Swa correlates with the less favorable entropy of binding of LC8 to Swa relative to IC. We propose that the conformational motility of beta 3 has functional significance in dimerization and in ligand binding. In the latter, beta 3 flexibility apparently accommodates different binding modes for different ligands resulting in ligand-specific conformational dynamics of the binding site that may impact other processes such as accessibility to phosphorylation.     

Glycosaminoglycans bind factor Xa in a Ca2+-dependent fashion and modulate its catalytic activity

Recent studies have demonstrated the existence of a Ca(2+)-dependent heparin-binding site on factor Xa. To characterize this heparin-binding site, the extrinsic fluorescence of fluorescein-labeled, active site-blocked factor Xa was monitored as it was titrated with glycosaminoglycans of various sulfate content and chain length. The binding of glycosaminoglycans to factor Xa appears to be charge-dependent because affinity is correlated with degree of glycosaminoglycan sulfation. All glycosaminoglycans bind factor Xa with higher affinity in the presence of Ca(2+) than in its absence. In contrast, when Gla-domainless factor Xa was substituted for factor Xa, glycosaminoglycans bound with similar affinities in the absence and presence of Ca(2+). These results support the hypothesis that the anionic Gla domain impairs glycosaminoglycan binding in the absence of Ca(2+). The changes in fluorescence intensity of factor Xa when titrated with glycosaminoglycans suggest that glycosaminoglycans induce conformational changes in the active site environment of factor Xa. To explore the consequences of these conformational changes, the effect of glycosaminoglycans on the catalytic activity of factor Xa was examined. Glycosaminoglycans influenced the ability of factor Xa to cleave chromogenic substrates and attenuated the capacity of factor Xa to activate factor VII. The potency of glycosaminoglycans in these assays reflected their affinity for factor Xa. These studies suggest that glycosaminoglycan binding perturbs exosites on the surface of factor Xa, potentially modifying interactions with cofactors or substrates.     

Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.     

Membrane interactions of a new class of anticancer agents derived from arylchloroethylurea: a FTIR spectroscopic study.

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and different lipids such as dimyristoylphosphatidylcholine (DMPC) in the absence and presence of 30 mol% of cholesterol, dimyristoylphosphatidylglycerol (DMPG) and a mixture made of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and DMPC by Fourier transform infrared (FTIR) spectroscopy. The results indicate that the drugs incorporate in the bilayer and cause a decrease of the phase transition temperature and an increase of the conformational disorder of the lipid acyl chains. These effects are dependent on the nature (degree of branching, length of the alkyl chain and presence of a sulfur atom), as well as on the position of the R substituent and are related to the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky branched substituent and those for which the disordering effect on the lipid bilayer is the greatest. On the other hand, the disordering effect is small for the long chain CEUs, such as 4-n-hexadecyl CEU, which have been shown to have weak cytotoxic activity.     

Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding.

To gain insight into the mechanism of facilitated sugar transport and possible mechanisms by which glucose transporter intrinsic activity might be altered, we have investigated conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding and by the transporter inhibitor, cytochalasin B. Changes in the ability of thermolysin to digest glucose transporters present in erythrocyte ghosts were used to monitor conformational changes of the glucose transporter. The degree of protease digestion was determined by the amount of undigested glucose transporter remaining after the protease treatment, as assessed in Western blots using the glucose transporter specific monoclonal antibody 7F7.5. D-Glucose, the physiological substrate of the transporter, increased the transporter's susceptibility to cleavage by thermolysin. Nontransportable glucose analogues which bind specifically to either an internal or external glucose transporter sugar binding site also altered susceptibility of the transporter to thermolysin. Both methyl and propyl glucoside, which preferentially bind the internal sugar site, increased thermolysin susceptibility of the glucose transporter in a manner similar to that of D-glucose. In contrast, 4,6-O-ethylideneglucose, which preferentially binds the external sugar site, protected the transporter from thermolysin digestion. These results suggest that sugar binding to internal and external sugar sites induces distinct conformational changes and that the observed D-glucose effect on the susceptibility of the glucose transporter to thermolysin is due to D-glucose at equilibrium predominantly forming a complex with the internal sugar site. The protection from cleavage by thermolysin caused by external sugar binding is attenuated by the addition of an internally binding sugar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and some properties of the Glu- and Lys-human plasmin heavy chains.

The heavy polypeptide chains of human Glu-plasmin and human Lys-plasmin have been isolated in native solvents, after partial reduction and carboxymethylation of the corresponding plasmins. Two major forms of each heavy chain can be eluted, after adsorption to Sepharose/lysine, utilizing a gradient of epsilon-aminocaproic acid as the eluant. The elution profile of these heavy chains is practically identical to the elution behavior previously observed for human Glu- and Lys-plasminogen, and human Glu- and Lys-plasmin adsorbed to these columns. Sedimentation velocity analysis of the heavy chain of human Glu-plasmin, in the presence of epsilon-aminocaproic acid, demonstrated that a gross conformational alteration occurs in this peptide accompanying binding of this amino acid. A much smaller conformational alteration occurs under similar circumstances with the human Lys-plasmin heavy chain. We find that the NH2-terminal peptide released in the Glu-plasminogen to Lys-plasminogen and Glu-plasmin to Lys-plasmin conversions is also released in the Glu-plasmin heavy chain to Lys-plasmin heavy chain conversion. This reaction is catalyzed at a significant rate only by plasmin and not by urokinase. Finally, no strong interaction between streptokinase and the isolated plasmin heavy chains is observed.     

Structure of beta-antithrombin and the effect of glycosylation on antithrombin's heparin affinity and activity

Antithrombin is a member of the serpin family of protease inhibitors and the major inhibitor of the blood coagulation cascade. It is unique amongst the serpins in that it circulates in a conformation that is inactive against its target proteases. Activation of antithrombin is brought about by a conformational change initiated upon binding heparin or heparan sulphate. Two isoforms exist in the circulation, alpha-antithrombin and beta-antithrombin, which differ in the amount of glycosylation present on the polypeptide chain; beta-antithrombin lacks the carbohydrate present at Asn135 in alpha-antithrombin. Of the two forms, beta-antithrombin has the higher affinity for heparin and thus functions as the major inhibitor in vivo even though it is the less abundant form. The reason for the differences in heparin affinity between the alpha and beta-forms have been shown to be due to the additional carbohydrate changing the rate of the conformational change. Here, we describe the most accurate structures of alpha-antithrombin and alpha-antithrombin+heparin pentasaccharide reported to date (2.6A and 2.9A resolution, respectively, both re-refinements using old data), and the structure of beta-antithrombin (2.6A resolution). The new structures have a remarkable degree of ordered carbohydrate and include parts of the antithrombin chain not modeled before. The structures have allowed a detailed comparison of the conformational differences between the three. They show that the structural basis of the lower affinity for heparin of alpha-antithrombin over beta-antithrombin is due to the conformational change that occurs upon heparin binding being sterically hindered by the presence of the additional bulky carbohydrate at Asn135.     

Development and validation of opioid ligand-receptor interaction models: the structural basis of mu vs delta selectivity.

Opioid receptor binding conformations for two structurally related, conformationally constrained tetrapeptides, JOM-6 ( micro receptor selective) and JOM-13 (delta receptor selective), were deduced using conformational analysis of these ligands and analogs with additional conformational restrictions. Docking of these ligands in their binding conformations to opioid receptor structural models, based upon the published rhodopsin X-ray structure, implicates specific structural features of the micro and delta receptor ligand binding sites as forming the basis for the micro selectivity of JOM-6 and the delta selectivity of JOM-13. In particular, the presence of E229 in the micro receptor (in place of the corresponding D210 of the delta receptor) causes an adverse electrostatic interaction with C-terminal carboxylate-containing ligands, resulting in the observed preference of ligands with an uncharged C-terminus for the micro receptor. In addition, the requirement that the Phe3 side chain of JOM-13 assume a gauche orientation for optimal delta binding, whereas the Phe3 side chain of JOM-6 must be in a trans orientation for high-affinity micro binding can be largely attributed to the steric effect of replacement of L300 of the delta receptor by W318 of the micro receptor. Testing this hypothesis by examining the binding of JOM-6 and several of its key analogs with specific micro receptor mutants is described. Our initial results are consistent with the proposed ligand-receptor interaction models.     

New evidence for the denaturant binding model.

Denaturation with guanidine hydrochloride (GdnHCl) or urea is one of the primary ways of measuring the conformational stability of proteins and comparing the stability of mutant proteins. Despite the widespread use of these two denaturants to provide quantitative data for the free energies of unfolding, the mode of action of these agents is not well understood. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or indirect and involves a change in the properties of solvent (water) in the presence of GdnHCl and urea. In this paper, an extensive kinetic study of the inhibition of ribonuclease A and papain by urea has been performed. The results suggest that the effect of urea on activities of these enzymes can be well described by the denaturant binding model. The binding constants of urea determined by the present method are nearly identical to that determined from a variety of different studies on model compounds and proteins.     

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Three dimensional structure of GD1b and GD1b-monolactone gangliosides in dimethylsulphoxide: a nuclear Overhauser effect investigation supported by molecular dynamics calculations.

A comparative study on the conformational features of the oligosaccharide moiety of GD1b and GD1b lactone gangliosides, in dimethylsulphoxide, has been carried out by nuclear Overhauser effect investigation; the experimental interresidue contacts have been used for restrained molecular mechanics and dynamics calculations. For GD1b, the tetrasaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2 ----8)-alpha-Neu5Ac-(2----3)]-beta-Gal has a circular arrangement leaving a highly hydrophobic region with seven hydrogens pointing towards the center. At one side of this region the three electron rich groups GalNAc--NH, external Neu5Ac--OH4 and internal Neu5Ac--COO- are grouped together; at the other side five polar groups (four hydroxy groups and the external Neu5Ac carboxylate) define a large annular hydrophilic region. The external Neu5Ac is close to the external Gal residue, and the external Neu5Ac--COO- is within van der Waals contact with the inner Neu5Ac-OH9 group. The beta-Gal-(1----3)-beta-GalNAc glycosidic linkage shows a high degree of freedom. For GD1b-L, the trisaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2----3)]-beta-Gal is disposed to forming rigid partially circular arrangement showing strong interresidue contacts between the inner Neu5Ac-H8 and both GalNAc-H1 and GalNAc-H5. The conformation of the lactone ring is the boat 9(A),2(B)B. The lactonization of the disialosyl residue induces a strong variation of the preexisting torsional glycosidic angles phi and psi, leaving the external Neu5Ac far from the external Gal. In both GD1b and GD1b lactone gangliosides, the conformation of the sialic acid side chain is the same as that of the free sialic acid in which the H7 is trans to H8 and gauche to H6, thus indicating that the presence of glycosidic and/or ester linkages does not affect the conformational properties of sialic acid. Both GD1b and GD1b lactone containing sialic acid carboxylate anion(s) or undissociated carboxyl group(s) show the same three dimensional structure, indicating that the presence of charges does not affect the intrinsic conformational features of gangliosides.     

Structure networks of E. coli glutaminyl-tRNA synthetase: effects of ligand binding

It is well known that proteins undergo backbone as well as side chain conformational changes upon ligand binding, which is not necessarily confined to the active site. Both the local and the global conformational changes brought out by ligand-binding have been extensively studied earlier. However, the global changes have been reported mainly at the protein backbone level. Here we present a method that explicitly takes into account the side chain interactions, yet providing a global view of the ligand-induced conformational changes. This is achieved through the analysis of Protein Structure Networks (PSN), constructed from the noncovalent side chain interactions in the protein. Here, E. coli Glutaminyl-tRNA synthetase (GlnRS) in the ligand-free and different ligand-bound states is used as a case study to assess the effect of binding of tRNA, ATP, and the amino acid Gln to GlnRS. The PSNs are constructed on the basis of the strength of noncovalent interactions existing between the side chains of amino acids. The parameters like the size of the largest cluster, edge to node ratio, and the total number of hubs are used to quantitatively assess the structure network changes. These network parameters have effectively captured the ligand-induced structural changes at a global structure network level. Hubs, the highly connected amino acids, are also identified from these networks. Specifically, we are able to characterize different types of hubs based on the comparison of structure networks of the GlnRS system. The differences in the structure networks in both the presence and the absence of the ligands are reflected in these hubs. For instance, the characterization of hubs that are present in both the ligand-free and all the ligand-bound GlnRS (the invariant hubs) might implicate their role in structural integrity. On the other hand, identification of hubs unique to a particular ligand-bound structure (the exclusive hubs) not only highlights the structural differences mediated by ligand-binding at the structure network level, but also highlights significance of these amino acids hubs in binding to the ligand and catalyzing the biochemical function. Further, the hubs identified from this study could be ideal targets for mutational studies to ascertain the ligand-induced structure-function relationships in E. coli GlnRS. The formalism used in this study is simple and can be applied to other protein-ligands in general to understand the allosteric changes mediated by the binding of ligands.     

Physicochemical properties of a novel Mr-21 000 Ca2+-binding protein of bovine brain.

The physicochemical properties of a novel Mr-21 000 Ca2+-binding protein isolated from bovine brain were investigated. The protein exhibited a partial specific volume of 0.724 ml/g, a degree of hydration of 0.47 g of water/g of protein and a mean residue weight of 119. Sedimentation equilibrium analysis revealed Mr = 22 600 in the absence of Ca2+; Ca2+ binding appeared to induce dimerization of the molecule. Size-exclusion chromatography indicated a compacting of the molecule on binding of Ca2+: the Stokes radius decreased from 2.75 nm in the absence of Ca2+ to 2.56 nm in its presence. Far-u.v.c.d. spectroscopy showed the apoprotein to be composed of 44% alpha-helix, 18% beta-pleated sheet and 38% random coil. Addition of either KCl (0.1 M) plus Mg2+ (1 mM), or Ca2+ (2 mM), changed the conformation to 49% alpha-helix, 18% beta-pleated sheet and 33% random coil. Near-u.v.c.d. and u.v. difference spectroscopy both indicated perturbations in the environments of all three types of aromatic amino acids on binding of Ca2+. Ca2+ binding also resulted in a 30% enhancement in the tryptophan fluorescence emission intensity. Ca2+ titration of the far-u.v.c.d. and fluorescence enhancement provided KD values of 9.91 microM and 4.68 microM respectively. Finally, the protein was shown to bind Zn2+ with KD = 1.44 microM (no Mg2+) and 1.82 microM (+ Mg2+). These observations strongly support the possibility that this novel Ca2+-binding protein resembles calmodulin and related Ca2+-binding proteins and undergoes a conformational change on binding of Ca2+ which reflects a physiological role in Ca2+-mediated regulation of brain function.     

Fluorescence studies on dopamine beta-monooxygenase: effects of salts, pH changes, metal-chelating agents and Cu2+

The intrinsic protein fluorescence of dopamine beta-monooxygenase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) has been characterized. The fluorescence is dominated by emission from tryptophans in a hydrophobic environment. Changes in the conformation of the enzyme induced by anions, pH changes, metal-chelating agents and Cu2+ have been determined. Conformational transitions induced by anions take place at concentrations between 0.05 and 0.2 M. Most anions give rise to a blue-shift, while ClO4- induces a red-shift of the emission spectrum. pH dependence of the protein fluorescence revealed a conformational change between pH 6.0 and 5.0. The interactions between dopamine beta-monooxygenase and seven different metal-chelating agents have been investigated using protein fluorescence, heat inactivation, and inhibition measurements. All the metal-chelating agents are able to remove the active-site copper as demonstrated by complete inhibition of enzyme activity, restoration of activity by the addition of copper, and the observation that the enzyme becomes more sensitive to heat inactivation in the presence of chelating agents, thus behaving similarly to the copper-free apoenzyme. The charge and size of the chelating agents are of importance for the reaction with the active-site copper, which is consistent with a mechanism for removal of the copper, including a ternary enzyme-copper chelating agent complex. By contrast, under turnover conditions in the presence of substrates, dissociation of the active-site copper and chelation of the free copper is a dominating mechanism. Three distinct conformations were characterized on the basis of the fluorescence spectra and the degree of quenching by Cu2+ and I-. For the copper-free apoenzyme a unique binding site for binding of the first copper was demonstrated by larger quenching of the protein fluorescence than for binding of additional copper.     

Influence of temperature on the conformation of canine plasminogen: an analytical ultracentrifugation and dynamic light scattering study

Plasminogen is known to undergo an extremely large conformational change when it binds ligands; the two well-established conformations are either closed (absence of external ligand) or open (presence of external ligand). We show here that plasminogen is more complicated than can be accommodated by a two-state, closed/open, model. Temperature changes induce large structural changes which can be detected with either dynamic light scattering or analytical ultracentrifugation. The temperature-induced changes are not related to the classical closed/open conformational change since both closed and open forms of the protein are similarly influenced. It appears as though the packing density of the protein increases as the temperature is raised. Over the range 4-20 degrees C, the Stokes' radius of the classical closed plasminogen goes from 4.7 to 4.2 nm, and that of the classical open form goes from 5.55 to 5.0 nm. These changes in packing can be rationalized if temperature change induces a large conformational change and if this is accompanied by a large change in hydration, by a change in solute binding, or by a change in the total void volume of the protein.     

Evidence for the "dock, lock, and latch" ligand binding mechanism of the staphylococcal microbial surface component recognizing adhesive matrix molecules (MSCRAMM) SdrG

Staphylococcus epidermidis is an opportunistic pathogen and a major cause of foreign body infections. The S. epidermidis fibrinogen (Fg)-binding adhesin SdrG is necessary and sufficient for the attachment of this pathogen to Fg-coated materials. Based largely on structural analyses of the ligand binding domain of SdrG as an apo-protein and in complex with a Fg-like peptide, we proposed that SdrG follows a "dock, lock, and latch" mechanism to bind to Fg. This binding mechanism involves the docking of the ligand in a pocket formed between two SdrG subdomains followed by the movement of a C-terminal extension of one subdomain to cover the ligand and to insert and complement a beta-sheet in a neighboring subdomain. These proposed events result in a greatly stabilized closed conformation of the MSCRAMM-ligand complex. In this report, we describe a biochemical analysis of the proposed conformational changes that SdrG undergoes upon binding to its ligand. We have introduced disulfide bonds into SdrG to stabilize the open and closed forms of the apo-form of the MSCRAMM. We show that the stabilized closed form does not bind to the ligand and that binding can be restored in the presence of reducing agents such as dithiothreitol. We have also used F?rster resonance energy transfer to dynamically show the conformational changes of SdrG upon binding to its ligand. Finally, we have used isothermic calorimetry to determine that hydrophobic interactions between the ligand and the protein are responsible for re-directing the C-terminal extension of the second subdomain required for triggering the beta-strand complementation event.