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PubMed1998

Activating a zymogen without proteolytic processing: mutation of Lys15 and Asn194 activates trypsinogen.

The zymogen and mature enzyme forms of trypsin-like serine proteases exhibit a wide range of activities. The prototypical trypsinogen-trypsin system is an example of a minimally active zymogen and a maximally active mature protease. The present work identifies several features of trypsinogen which govern its activity. Our results indicate that rat trypsin is 10(8)-fold more active than rat trypsinogen. Rat trypsinogen appears to be less active than bovine trypsinogen. His40 is believed to be an important determinant of zymogen activity. We are unable to verify this role for His40 in trypsinogen since the mutation of His40 to Phe appears to change the trypsin-substrate interface. Deletion of the N-terminal Ile16 from trypsin is expected to produce a trypsinogen-like protein since the Ile16-Asp194 salt bridge cannot form. Such mutants have higher activity and BPTI affinity than trypsinogen, which indicates that the activation peptide stabilizes the inactive trypsinogen conformation. The mutation of Lys15 to Ala increases the BPTI affinity and activity of trypsinogen to an even greater extent; thus, removal of Lys15 can account for the effect of the loss of the activation peptide. These results suggest that Lys15 is an important determinant of zymogen activity. The mutation of Asp194 to Asn also increases the BPTI affinity and activity of trypsinogen. This result suggests that in addition to stabilizing the active conformation of trypsin via the Ile16-Asp194 salt bridge, Asp194 also maintains the inactive conformation of trypsinogen. A correlation exists between the values of kcat/Km and BPTI affinity of mutant trypsinogens and trypsins. However, the slope of this correlation is 0.64, which indicates that different "active" conformations are involved in BPTI binding and substrate hydrolysis. DeltaI16V17 trypsinogen is the lone outlier; its BPTI affinity is higher than would be expected based on the value of kcat/Km. We show that the rate of BPTI association is slower for DeltaI16V17 trypsinogen than for a mutant trypsinogen with a similar BPTI affinity. This observation suggests that BPTI binds to an "active" trypsinogen conformation that is not kinetically accessible to substrates.

Animals↗
PubMed2003

Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila.

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.

Amino Acids↗
PubMed1997

A molecular switch for biochemical logic gates: conformational studies.

This report presents the computer-assisted design of a molecular switching element, in which a molecular switch regulates the enzymatic activity of Ribonuclease A (RNase A). The molecular switch, an appropriately modified amino acid residue, is constructed with an electron donor group and an electron acceptor group, connected to one another with a conjugated double bond bridge. The switching mechanism is based on the azonium-hydrazo tautomerization, by which a charge separation induced in the excited state causes a rearrangement of the molecular electronic structure, resulting in the exchange of locations of single and double bonds. This rearrangement of bonds leads to different three-dimensional conformations of the switch. Using the electrostatically driven Monte Carlo (EDMC) method and the empirical conformational energy program for peptides (ECEPP/3) potential energy function, we carried out an exhaustive search of the conformational space of the switching element. The results of these calculations reveal two sets of conformations: in one set the access to the active site of the enzyme is preferentially blocked, while in the other set the active site is preferentially accessible. Integration of the designed element into biochemical logic gates operating under the rules of threshold value, and experimental implementation of this system, are considered.

Protein Conformation↗
PubMed1997

Desensitization of the nicotinic acetylcholine receptor mainly involves a structural change in solvent-accessible regions of the polypeptide backbone.

The difference between infrared spectra of the nicotinic acetylcholine receptor (nAChR) recorded using the attenuated total reflectance technique in the presence and absence of carbamylcholine exhibits a complex pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon agonist binding and subsequent desensitization. Two relatively intense bands are observed in the amide I region of the difference spectra recorded in 1H2O buffer near 1655 cm(-1) and 1620 cm(-1) that were previously interpreted in terms of either a net conversion of beta-sheet to alpha-helix or a reorientation of transmembrane alpha-helix accompanied by a change in structure of beta-sheet and/or turn [Baenziger, J. E., Miller, K. W., & Rothschild, K. J. (1993) Biochemistry 32, 5448-5454]. However, difference spectra recorded in 2H2O buffer reveal that these and other difference bands in the amide I region undergo downshifts in frequency upon peptide 1H/2H exchange that are much larger than the downshifts in frequency that are typically observed for the amide I vibrations of either alpha-helix or beta-sheet. Difference spectra recorded in 2H2O buffer within either minutes or hours of prior exposure of the nAChR to 2H2O exhibit the same amide I difference band shifts that are observed in difference spectra recorded after 3 days prior exposure of the nAChR to 2H2O. Most of the peptides that are involved in both ligand binding and the resting to desensitized conformational change and that give rise to bands in the difference spectra therefore exchange their hydrogens for deuterium on the seconds to minutes time scale. The frequencies of the difference bands, the magnitudes of the difference band shifts upon peptide 1H/2H exchange, and the rapidity of the hydrogen deuterium exchange kinetics of those structures that give rise to amide I bands in the difference spectra all suggest that the formation of a channel-inactive desensitized state results predominantly from a conformational change in solvent-accessible extramembranous regions of the polypeptide backbone as opposed to a large structural perturbation near the ion channel gate. A conformational change in the agonist binding site may be primarily responsible for channel inactivation upon desensitization.

Carbachol↗
PubMed2006

An inward-facing conformation of a putative metal-chelate-type ABC transporter.

The crystal structure of a putative metal-chelate-type adenosine triphosphate (ATP)-binding cassette (ABC) transporter encoded by genes HI1470 and HI1471 of Haemophilus influenzae has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B12 importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.

ATP-Binding Cassette Transporters↗
PubMed2006

Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3.

Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.

Bacterial Proteins↗
PubMed1992

Minimum energy conformations of proline-containing helices.

Proline occurs frequently in transmembrane alpha-helices of transport and receptor proteins even though statistical surveys demonstrate the overwhelming preference of this residue for a non-alpha-helical, hydrophilic environment. As a result, membrane-buried proline has been proposed to be functionally important, with function arising from structural discontinuity or destabilization of the helix. Destabilization may occur by Pro-mediated conformational transitions between discrete states, and may be manifested in membrane protein systems through reversible processes such as channel opening and closing or signal transduction. In this study, computer modeling of a model transmembrane alpha-helix, (Ala)8-Leu-Pro-Phe-(Ala)8, in a medium of low polarity (dielectric = 2), is used to examine the occurrence and energetic accessibility of Pro-mediated conformational interconversions. Leu psi and chi 1, Pro psi, and Phe phi and chi 1 torsion angles were assigned random values so that a data base of 200 conformations for each of the cis and trans states was generated. The conformations were minimized and low-energy structures organized into families. This analysis demonstrated that the most populated lowest energy family is the Trans-I conformation, corresponding to proline in a kinked alpha-helix. Two additional trans structures, Trans-II and Trans-III, as well as a cis conformation, Cis-I, are also energetically competitive. Interconversions between the trans states could thus be mediated by changes at a single torsion angle, accompanied by minor local hydrogen-bonding rearrangements. This work substantiates that membrane-buried proline can provide the basis for conformational transitions between discrete alpha-helix-based structures in a nonpolar environment.

Amino Acid Sequence↗
PubMed1993

Conformational changes in oxidized phospholipids and their preferential hydrolysis by phospholipase A2: a monolayer study.

Cleavage of oxidized fatty acids by phospholipase A2 has been implicated as the first step in the repair mechanism for oxidative damage to membrane phospholipids. However, the mechanism by which this enzyme preferentially hydrolyzes oxidized fatty acyl chains is poorly understood. Using a lipid monolayer technique, we found that the molecular surface areas of 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)-phosphatidylcholine (PLPC-OOH) and 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) were increased by as much as 50% relative to the parent nonoxidized 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC). These experimental data directly indicate a drastically changed molecular conformation of oxidized phospholipids in which the hydroperoxy or hydroxy group in the sn-2 fatty acid is close to the lipid-water interface. Phospholipases A2 from porcine pancreas and from bee venom were shown to break down PLPC-OOH and PLPC-OH monolayers much faster than PLPC monolayers. In all cases, the presence of serum albumin in the subphase enhanced monolayer breakdown by extracting hydrolysis products from the monolayer, but monolayer breakdown was always much faster for oxidized than for nonoxidized PLPC. This did not appear to be due to change in the extent of monolayer penetration by phospholipase A2, since enzyme-monolayer interaction studies revealed essentially identical penetration behavior of bee venom phospholipase A2 with PLPC, PLPC-OOH, and PLPC-OH monolayers. We propose that the altered molecular conformation of oxidized phospholipids facilitates access to the sn-2 ester bond, thereby ensuring their preferential hydrolysis in the presence of a phospholipase A2.

Hydrolysis↗
PubMed1999

Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase.

Activation of the superoxide-producing phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox), which is mediated via the SH3 domains of the former protein. This interaction is considered to be induced by exposure of the domains that are normally masked by an intramolecular interaction with the C-terminal region of p47(phox). Here we locate the intramolecular SH3-binding site at the region of amino acid residues 286-340, where Ser-303, Ser-304, and Ser-328 that are among several serines known to become phosphorylated upon cell stimulation exist. Simultaneous replacement of the three serines in p47(phox) with aspartates or glutamates, each mimicking phosphorylated residues, is sufficient for disruption of the intramolecular interaction and resultant access to p22(phox). The triply mutated proteins are also capable of activating the NADPH oxidase without in vitro activators such as arachidonate under cell-free conditions. In a whole-cell system where expression of the wild-type p47(phox) reconstitutes the stimulus-dependent oxidase activity, substitution of the kinase-insensitive residue alanine for Ser-328 as well as for Ser-303/Ser-304 leads to a defective production of superoxide. These findings suggest that phosphorylation of the three serines in p47(phox) induces a conformational change to a state accessible to p22(phox), thereby activating the NADPH oxidase.

Amino Acid Substitution↗
PubMed2000

Crystallographic analysis of protein conformational changes in the bacteriorhodopsin photocycle.

A variety of neutron, X-ray and electron diffraction experiments have established that the transmembrane regions of bacteriorhodopsin undergo significant light-induced changes in conformation during the course of the photocycle. A recent comprehensive electron crystallographic analysis of light-driven structural changes in wild-type bacteriorhodopsin and a number of mutants has established that a single, large protein conformational change occurs within 1 ms after illumination, roughly coincident with the time scale of formation of the M(2) intermediate in the photocycle of wild-type bacteriorhodopsin. Minor differences in structural changes that are observed in mutants that display long-lived M(2), N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. These observations support a model for the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M(1)) are well approximated by one protein conformation in which the Schiff base has extracellular accessibility, while the structures of the later intermediates (M(2), N and O) are well approximated by the other protein conformation in which the Schiff base has cytoplasmic accessibility.

Bacteriorhodopsins↗
PubMed2002

Side-chain conformational entropy at protein-protein interfaces.

Protein-protein interactions are the key to many biological processes. How proteins selectively and correctly associate with their required protein partner(s) is still unclear. Previous studies of this "protein-docking problem" have found that shape complementarity is a major determinant of interaction, but the detailed balance of energy contributions to association remains unclear. This study estimates side-chain conformational entropy (per unit solvent accessible area) for various protein surface regions, using a self-consistent mean field calculation of rotamer probabilities. Interfacial surface regions were less flexible than the rest of the protein surface for calculations with monomers extracted from homodimer datasets in 21 of 25 cases, and in 8 of 9 for the large protomer from heterodimer datasets. In surface patch analysis, based on side-chain conformational entropy, 68% of true interfaces were ranked top for the homodimer set and 66% for the large protomer/heterodimer set. The results indicate that addition of a side-chain entropic term could significantly improve empirical calculations of protein-protein association.

Binding Sites↗
PubMed1998

Enzymatic and structural similarities between the Escherichia coli ATP-dependent proteases, ClpXP and ClpAP.

Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (Mr 46, 000) associate to form a six-membered ring (Mr approximately 280, 000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (approximately 2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min-1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

[Effect of surfactants on peroxidase activity. I. Effect of anionic surfactants].

The effect of various anionic surfactants on the initial rate of 5-aminosalicylic acid peroxidation with horseradish peroxidase was studied. With increasing surfactant concentration, this rate first decreased, then increased, and again decreased. We conclude that these changes were due to an inhibition of the enzyme, a subsequent change in the enzyme conformation accompanied by the enhanced accessibility of its active site to the substrate, and the final protein denaturation, respectively.

Anions↗
PubMed2002

Binding and release of iron by gel-encapsulated human transferrin: evidence for a conformational search.

Human transferrin is a single-chain bilobal protein with each of the two similar but not identical lobes in turn composed of two domains. Each lobe may assume one of two stable structural conformations, open or closed, determined by a rigid rotation of the domains with respect to each other. In solution, the transformation of a lobe between open and closed conformations is associated with the release or binding of an Fe(III) ion. The results of the present study indicate that encapsulation of transferrin within a porous sol-gel matrix allows for a dramatic expansion, to days or weeks, of this interconversion time period, thus providing an opportunity to probe heretofore inaccessible transient intermediates. Sol-gel-encapsulated iron-free transferrin samples are prepared by using two protocols. In the first protocol, the equilibrium form of apotransferrin is encapsulated in the sol-gel matrix, whereas in the second protocol holotransferrin is first encapsulated and then iron is removed from the protein. Results of kinetic and spectroscopic studies allow for distinguishing between two models for iron binding. In the first, iron is assumed to bind to amino acid ligands of one domain, inducing a rigid rotation of the second domain to effect closure of the interdomain cleft. In the second, iron undertakes a conformational search among the thermally accessible states of the lobe, "choosing" the state which most nearly approximates the stable closed state when iron is bound. Our experimental results support the second mechanism.

Apoproteins↗
PubMed1993

Hydroxyl radical footprint analysis of human immunodeficiency virus reverse transcriptase-template.primer complexes.

Human immunodeficiency virus type 1 reverse transcriptase protects sugar moieties of a model template.primer DNA in a region from positions +3 to -15 from hydroxyl radical attack. A protected region of equivalent size migrates in concert with the translocating enzyme, as shown by hydroxyl radical footprints of replication complexes after primer extension by 4, 10, and 19 nt. The pattern of these footprints suggests that the DNA template.primer is in the A conformation when complexed with reverse transcriptase. Enhanced accessibility of the DNA template strand around position -15 to hydroxyl radicals indicates a conformational change in the template induced by the C-terminal RNase H-containing domain of p66 reverse transcriptase.

Base Sequence↗
PubMed1992

Common and different antigenic properties of the rabies virus glycoprotein of strains SAD-Vnukovo and Pitman-Moore.

Two fixed rabies virus strains, SAD-Vnukovo and Pitman-Moore (PM) were used as combined immunogens for the generation of hybridomas secreting specific monoclonal antibodies (MoAbs). The obtained hybridomas were primarily screened by an ELISA for production of MoAbs to antigen of SAD-Vnukovo strain. Six positive clones were established. A panel of MoAbs has been characterized according to reactivity in immunofluorescence, immunoblot, ELISA and neutralization tests. All MoAbs were positive in immunofluorescence when cells infected with the SAD-Vnukovo strain were used. By immunoblot, four MoAbs showed specificity for the viral glycoprotein of both SAD-Vnukovo and PM rabies strains. This pattern of reactivity indicated the existence of shared conformation-independent epitopes located on the related antigens. However, in ELISA, the tested MoAbs did not recognize viral glycoproteins of the PM strain. This indicates, that the different strain-specific conformations of the native glycoprotein determine the accessibility of the common linear determinants for respective antibodies. Only one antibody, with conformation-dependent glycoprotein specificity, was capable to neutralize the CVS strain of rabies virus.

Animals↗
PubMed2005

Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al. (2005) Arch. Biochem. Biophys. 433, 297-311]. The open conformation is important for substrate binding and product release, the in conformation for reaction with oxygen and hydroxylation, and the out conformation for the reduction of FAD by NADPH. The open conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the si side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the in conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the si side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1998

Folding of firefly (Photinus pyralis) luciferase: aggregation and reactivation of unfolding intermediates.

The guanidine-induced unfolding of firefly (Photinus pyralis) luciferase involves two inactive equilibrium intermediates and is freely reversible at low protein concentration and low temperature. However, reactivation is exceedingly slow so that the equilibrium is attained only after several days of incubation and reactivation yields decrease strongly with increasing protein concentration, suggesting that aggregation is a competing side reaction [Herbst et al. (1997) J. Biol. Chem. 272, 7099-7105]. We investigated the role of the equilibrium intermediates in the aggregation process using size-exclusion chromatography and dynamic light scattering to monitor their association state. Although the more unfolded intermediate aggregated much more rapidly, both intermediates associated irreversibly without a conformational change visible by fluorescence or circular dichroism, forming small oligomers which remained soluble in the presence of the denaturant. The association kinetics are compatible with a nucleated polymerization mechanism. Unfolding kinetics at 1 M denaturant indicated the presence of a further inactive intermediate capable to reactivate rapidly with kinetics similar to those observed for luciferase reactivation in the presence of cell extracts. The data suggest a kinetic trap in luciferase refolding that is accessible from both equilibrium intermediate conformations and is avoided in the presence of molecular chaperones.

Animals↗