Conformational Motility Research Articles

The Dynamics of OXA-23 β-Lactamase from Acinetobacter baumannii.

Antibiotic resistance is a pressing topic, which also affects β-lactam antibiotic molecules. Until a few years ago, it was considered no more than an interesting species from an academic point of view, Acinetobacter baumanii is today one of the most serious threats to public health, so much so that it has been declared one of the species for which the search for new antibiotics, or new ways to avoid its resistance, is an absolute priority according to WHO. Although there are several molecular mechanisms that are responsible for the extreme resistance of A. baumanii to antibiotics, a class D β-lactamase is the main cause for the clinical concern of this bacterial species. In this work, we analyzed the A. baumanii OXA-23 protein via molecular dynamics. The results obtained show that this protein is able to assume different conformations, especially in some regions around the active site. Part of the OXA-23 protein has considerable conformational motility, while the rest is less mobile. The importance of these observations for understanding the functioning mechanism of the enzyme as well as for designing new effective molecules for the treatment of A. baumanii is discussed.

Photo-induced conformational motility of proteins

The dynamics of proteins, detected by fluorescence, consists of three components: spontaneous dynamics, dipole-dipole photo-induced dynamics, thermal photo-induced dynamics. The photo-induced dynamics can lead to activation as well as inactivation of enzymes.

Conformational studies of the robust 2-Cys peroxiredoxin Salmonella typhimurium AhpC by solution phase hydrogen/deuterium (H/D) exchange monitored by electrospray ionization mass spectrometry

This is the first comprehensive HX-MS study of a “robust” 2-Cys peroxiredoxin (Prx), namely Salmonella typhimurium AhpC (StAhpC). Prx proteins control intracellular peroxide levels and are abundant antioxidant proteins in eukaryotes, archaea and bacteria blue. Crystal structural analyses and structure/activity studies of several bacterial and mammalian 2-Cys Prxs have revealed that the activity of 2-Cys Prxs is regulated by redox-dependent oligmerization and a sensitivity of the active site cysteine residue to overoxidation. The propensity to overoxidation is linked to the conformational flexibility of the peroxidatic active site loop. The HX-MS results emphasize the modulation of the conformational motility of the active site loop by disulfide formation. To obtain information on the conformational impact of decamer formation on the active site loop motility, mutants with Thr77 substituted by Ile, a decamer-disrupting mutation or by Val, a decamer-stabilizing mutation, were studied. For the isoleucine mutant, enhanced mobility was observed for regions encompassing the α4 helix located in the dimer–dimer interface and regions surrounding the peroxidatic loop. In contrast, the T77V mutation resulted in an increase in conformational stability in most regions of the protein except for the active site loop and the region encompassing the resolving cysteine.

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.

Conformational analysis of cyclic dipeptides: I. , , , and

Conformational analysis of two pairs of synthetic cyclodipeptides formed by interaction of both side chain functional groups [Formula: see text] and of the main and side chains [Formula: see text] was achieved by the method of molecular mechanics. The energetically optimal conformational states of the molecules under study were determined. It was shown that the conformational motility of cyclic system of the compounds under study depends on the relative arrangement of the amide groups and the number of atoms in the cycle.

Measuring enzyme motility in organic media using novel H‐D exchange methodology

A novel deuterium (2H) NMR technique as developed for measuring the total number of deuterons exchanged by lyophilised protein samples following hydrogen-deuterium (H-D) exchange. Using this methodology differences in the H-D exchange behaviour of the proteolytic enzyme subtilisin Carlsberg hydrated either in air or an organic solvent were probed as a function of hydration. At low thermodynamic water activity (aw), the degree of H-D exchange increased rapidly with hydration (from anhydrous to aw 0.22). At aw 0.22, subtilisin powders hydrated in air were found to have reached an H-D exchange level comparable to that found upon aqueous dissolution and in agreement with previous studies using lysozyme. Lyophilised subtilisin hydrated in either dichloromethane (DCM) or diisopropyl ether (DIPE) showed a pattern of exchange (vs. aw) comparable to that found for powders hydrated in air. However, subtilisin hydrated in n-hexane showed a significant reduction in H-D exchange at all aw studied. Control experiments demonstrated that the reduction in H-D exchange observed for subtilisin in n-hexane was not a kinetic effect. This lower level of exchange in n-hexane implies that hydrated subtilisin Carlsberg has a lower conformational motility and more rigid protein matrix. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 262–269, 2000.

Measuring enzyme motility in organic media using novel H-D exchange methodology

A novel deuterium ((2)H) NMR technique as developed for measuring the total number of deuterons exchanged by lyophilised protein samples following hydrogen-deuterium (H-D) exchange. Using this methodology differences in the H-D exchange behaviour of the proteolytic enzyme subtilisin Carlsberg hydrated either in air or an organic solvent were probed as a function of hydration. At low thermodynamic water activity (a(w)), the degree of H-D exchange increased rapidly with hydration (from anhydrous to a(w) 0.22). At a(w) 0.22, subtilisin powders hydrated in air were found to have reached an H-D exchange level comparable to that found upon aqueous dissolution and in agreement with previous studies using lysozyme. Lyophilised subtilisin hydrated in either dichloromethane (DCM) or diisopropyl ether (DIPE) showed a pattern of exchange (vs. a(w)) comparable to that found for powders hydrated in air. However, subtilisin hydrated in n-hexane showed a significant reduction in H-D exchange at all a(w) studied. Control experiments demonstrated that the reduction in H-D exchange observed for subtilisin in n-hexane was not a kinetic effect. This lower level of exchange in n-hexane implies that hydrated subtilisin Carlsberg has a lower conformational motility and more rigid protein matrix.

Altering the state of phosphorylation of rat liver keratin intermediate filaments by ethanol treatment in vivo changes their structure

Dephosphorylation of keratin intermediate filaments (IF) in livers from ethanol-fed rats relative to controls occurs concurrently with a reorganization of the distribution of IF in the cells. One possible molecular mechanism for this reorganization is a phosphorylation-induced conformational change in the keratin that propagates as a change in the polymerization of the keratin subunits. To test this hypothesis, the structure of liver keratin IF, from both control and alcohol-fed rats, was explored by circular dichroism (CD), tryptophan fluorescence quenching, and 13C nuclear magnetic resonance (NMR). Keratin IF were isolated from livers of control rats and from livers of rats that had ethanol included in their feed for 6–40 weeks. A significant decrease in the intensity of the CD spectrum of keratin IF from livers of ethanol-treated animals, relative to controls, was observed. These data suggested either that a change in conformation or an increase in conformational motility in the keratin IF from ethanol-treated animals occurred as a result of the ethanol-induced dephosphorylation. 13C NMR data were obtained to distinguish between these two possibilities. An increase in resonance intensity of some 13C NMR resonances was observed in the keratin IF from livers of ethanol-treated animals, relative to controls. The CD and NMR data were therefore consistent with an increase in conformational motility of the rod domain in these keratin IF. No significant change was observed in the quenching of tryptophan fluorescence by KI. The change in protein dynamics detected in these experiments could be the molecular basis for the alteration of keratin IF organization in alcoholic hepatitis.

A family of novel peroxidases, peroxiredoxins and receptor signaling

This is the first comprehensive HX-MS study of a “robust” 2-Cys peroxiredoxin (Prx), namely Salmonella typhimurium AhpC (StAhpC). Prx proteins control intracellular peroxide levels and are abundant antioxidant proteins in eukaryotes, archaea and bacteria blue. Crystal structural analyses and structure/activity studies of several bacterial and mammalian 2-Cys Prxs have revealed that the activity of 2-Cys Prxs is regulated by redox-dependent oligmerization and a sensitivity of the active site cysteine residue to overoxidation. The propensity to overoxidation is linked to the conformational flexibility of the peroxidatic active site loop. The HX-MS results emphasize the modulation of the conformational motility of the active site loop by disulfide formation. To obtain information on the conformational impact of decamer formation on the active site loop motility, mutants with Thr77 substituted by Ile, a decamer-disrupting mutation or by Val, a decamer-stabilizing mutation, were studied. For the isoleucine mutant, enhanced mobility was observed for regions encompassing the α4 helix located in the dimer–dimer interface and regions surrounding the peroxidatic loop. In contrast, the T77V mutation resulted in an increase in conformational stability in most regions of the protein except for the active site loop and the region encompassing the resolving cysteine.

Renal brush border membrane lipid composition in Basenji dogs with spontaneous idiopathic Fanconi syndrome

To comprehend the renal defect underlying idiopathic Fanconi syndrome in the Basenji dog, we have focused on delineating the lipid profiles of renal brush border membranes isolated from affected and normal Basenji dogs to establish any physical or compositional changes underlying previously observed transport and membrane-fluidity changes. The lipid composition was studied with respect to total lipid, cholesterol, and phospholipid content, cholesterol to phospholipid ratio, distribution of the major phospholipid classes, and fatty acid composition. Total phospholipid of the isolated renal brush border membranes from Fanconi syndrome dogs analyzed by 31P nuclear magnetic resonance showed no difference compared with that of normal dogs. Examination of total fatty acids in both membranes using gas-liquid chromatography analysis of fatty acid methyl esters showed no difference in the mole percents of the major fatty acids. Our data suggest that changes in bulk membrane fluidity of the Fanconi syndrome dog renal brush border as measured by 1,6-diphenyl-1,3,5-hexatriene cannot be attributed to phospholipid and fatty acid compositional change. In the membranes isolated from affected dog kidney, the cholesterol content determined by gas-liquid chromatography analysis was 66 mol% higher than in membranes isolated from normal dog kidney. This correlates with the higher cholesterol to phospholipid molar ratio of 0.82 ± 0.08 in the affected animal as compared with 0.58 ± 0.04 in the normal. Cholesterol content and its microdomain in the membrane bilayer may be important in modulating transport functions. Increased membrane cholesterol content may affect the conformational motility of membrane transport proteins and thus affect their function.

Conformational changes in biological macromolecules.

Some known conformational changes occurring in proteins and peptides are summarized and discussed. A novel nonradiative energy transfer technique, enabling evaluation of the conformation and intramolecular motility of polypeptides in solution, is described. The method should prove useful in the elucidation of the conformational motility prevailing in various biopolymers. The following three possible levels of flexibility in proteins are described: a. flexibility of chain segments; b. domain flexibility; and c. motion of side chains. Evidence is forwarded to show that these conformational motions actually occur in some proteins, as revealed by a number of physical chemical techniques. The possible biological significance of the predicted conformational flexibility and conformational fluctuations of biopolymers is finally discussed.

Comparative study of the conformational transitions of frog and rabbit phosphorylases B

1. 1. Conformational motility of the purified muscle glycogen phosphorylase B from two species of vertebrates (rabbit and frog) was investigated by the Hydrogen-Exchange method and Infrared Spectrometry. 2. 2. The experimental results of the 1H- 2H exchange were expressed interms of the probability of P of exposure to isotopic solvent of phosphorylase peptide groups and in terms of the corresponding changes in standard free energy ΔG 0. 3. 3. The combined methods used didn't show considerable differences of the protein conformations in the physiological pH region but rabbit phosphorylase was only characterized by rather more compact structure in comparison with frog phosphorylase.

Increase of conformational stability of homogeneous rabbit immunoglobulin g after hapten binding

Hydrogen-deuterium exchange, adiabatic scanning microcalorimetry and difference sedimentation were used to detect ligand-induced conformational changes in a homogeneous rabbit antibody. Site-filling monovalent and bivalent ologosaccharide ligands, derived from the type III pneumococcal polysaccharide, were used. Only those antigen-antibody complexes which were monomeric with respect to antibody were included into this study. Hydrogen-deuterium exchange reflects the probability of solvent exposure of peptide hydrogens. In this study, the comparative analysis of the probability distributions for various antibody-ligand complexes demonstrates that most if not all of the observable exchange rates were affected by the bincling of hapten. Scanning microcalorimetry detects changes in the overall conformational stability of the antibody molecule. Biphasic heat absorption curves were obtained, reflecting biphasic heat-induced conformational transitions. The significant hapten-induced shift in the temperature of the first transition was interpreted as a consequence of tightening in the domain-domain interactions, while the slight shift in the second transition can be explained by a stabilization of the domains themselves. No difference could be detected in the hydrodynamic properties of the liganded and the unliganded antibodies by difference sedimentation. The effect of monovalent or bivalent hapten bincling upon antibody conformation manifests itself in restricted conformational motility and enhanced conformational stability, without any detectable change in the shape or the volume of the molecule.

Conformation of gamma-crystallins of the calf lens: Effects of temperature and denaturing agents

The secondary and tertiary conformations of calf γ-crystallin were monitored by circular dichroism (CD) measurements in the near and far ultraviolet regions. The secondary structures of the various fractions of γ-crystallin do not possess any α-helix conformation as judged by the far ultraviolet CD spectra. Each of the various γ-crystallin fractions exhibited a unique and complex near ultraviolet CD spectrum, reflecting the contributions of tryptophanyl, tyrosyl and phenylalanyl residues to the optical activity. The low molecular weight β-crystallin fraction, β s, possesses its own characteristic secondary and tertiary structural parameters. β s-crystallin contains about 10% of α-helical structure and its CD wavelength profile in the near ultraviolet is different from that observed for the various γ-crystallin fractions. One per cent sodium dodecyl sulfate and 5 m-guanidine hydrochloride were very effective in the disruption of the secondary and tertiary structure of γ-crystallin as judged from the changes observed in the CD spectra. On the other hand, 8 m-urea caused only minor perturbation in the CD wavelength profile, suggesting that γ-crystallin did not denature at high urea concentration. The conformation of γ-crystallin as a function of temperature was monitored by CD measurements between −20°C and +60°C at pH values of 5·5 and 8·5 and at various protein concentrations ranging from 1 to 40 mg/ml. No significant spectroscopic changes were observed within this temperature range. The independence of the CD spectra from temperature suggests that the aromatic residues of γ-crystallin are in a rigid environment and do not possess appreciable conformational motility. Previous work Zigman and Lerman 1965 has suggested that γ-crystallin (which was classified as a “cryoprotein”) when cooled, changes its conformation in such a way as to cause precipitation. While conformational changes as a function of temperature could be detected by optical measurements in other cryoproteins, we have failed to detect such changes in γ-crystallins. The fact that γ-crystallin precipitates at relatively high concentration in a very narrow pH range as the temperature is lowered, can simply be explained as isoelectric precipitation.

Substrate-induced deactivation of penicillinases. Studies of beta-lactamase I by hydrogen exchange.

The conformational motility of beta-lactamase I from Bacillus cereus was studied by hydrogen exhange. The time course of the isotopic replacement of peptide hydrogen atoms was followed by 'exchange-in' or 'exchange-out' experiments. Many of the substrates for this enzyme that have o-substituted aromatic or heterocyclic side chains (e.g. methicillin or cloxacillin) are known to effect a decrease in enzymic activity ('substrate-induced deactivation'). There was a marked discontinuity in the exchange-out curve when methicillin or cloxacillin was diffused into the enzyme solution. About one-half of the hydrogen atoms that were probed were affected by the presence of these substrates, and the change in the reactivity of the hydrogen atoms was also large. Substrates that do not bring about deactivation (benzylpenicillin and cephalosporin C) do not affect the hydrogen exchange, nor do reversible competitive inhibitors such as the penicilloic acid or penilloic acid. On the other hand, Zn2+ ions do affect the hydrogen exchange; their effect is similar to that of methicillin or cloxacillin.

Effect of ligand and heme on conformational stability (intramolecular conformational motility) of hemoglobin as revealed by hydrogen exchange

Recently published crystallographic data show that functionally important events are frequently accompanied by small, in absolute magnitude, but widespread perturbation of protein structure [l-5]. These conformational rearrangements are still hardly enlightened because they do not usually affect the content of regular structure, the fraction of solvent, exposed aromatic residues or the shape of molecule and therefore could not be detected by optical or hydrodynamic methods. To clarify the physical basis and the functional meaning of such subtle conformational changes it is necessary to know whether they lead to any changes in conformational energy of the protein. To study this we have chosen two derivatives of Hb MetHbHzO and MetHbCN as a model because recently the above-mentioned conformational differences between these two liganded Hbs have been well documented by a sensitive difference Fourier

Immunological measurements of conformational motility in regions of the myoglobin molecule

The conformational motilities of three regions of the sperm whale myoglobin molecule and of an isolated peptide of myoglobin have been examined by measuring the equilibrium constant for the native equilibrium nonnative transition. The immunological approach of Furie et al. (Furie, B., Schechter, A.N., Sachs D., and Anfinsen, C.B. (1975), J. Mol. Biol.92, 497-506) was used with convenient modifications. Antibodies specific to the nonnative conformations were used in assaying for competition between the radioactively labeled peptide and native myoglobin. Labeling was by 125I iodination of the peptide or its 3-(4-hydroxyphenyl)propionyl derivative, and separation of the immune complex from the free peptide was either by ammonium sulfate precipitation or by centrifugation of the antibodies immobilized on Agarose beads. For the antigenic regions of the sequence (1-55), the measured conformational equilibrium constant was 840 +/- 200 at 22 degrees C; the value for the C-terminal region (132-153) was 280 +/- 120 at 25 degrees C, while that for the region (66-76) adjacent to the heme group was greater than 2.5 x 10(6). Measurements on the isolated peptide (132-153) indicated that 1% of the molecules adopt native-type folding in aqueous solution at 36 degrees C.

Effect of inter-subunit contact on intramolecular conformational motility (conformational stability) of hemoglobin as revealed by hydrogen exchange

Effect of inter-subunit contact on intramolecular conformational motility (conformational stability) of hemoglobin as revealed by hydrogen exchange

The role of interchain disulphide bridges in the conformational stability of human immunoglobulin G1 subclass. Hydrogen-deuterium exchange studies.

The hydrogen-deuterium exchange data of human immunoglobulin G1 (IgG1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10(-3) for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol). In the range of pH 6.2-8.45, at 25 degrees C no gross conformational changes are reflected in the hydrogen-deuterium exchange behaviour of the native, the reduced-nonalkylated-reassociated and the reduced-S-alkylated-reassociated IgG1. No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter-subunit disulphide bridges in S-alkylated-reassociated molecules results in an increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average.

Unpaired bases in superhelical DNA: kinetic evidence.

Kinetic analysis of the early, fast reaction of superhelical DNA with formaldehyde reveals that this region or regions is 56% "single strand like" in character. Hydrogen-tritium exchange studies coupled with other considerations show that this reaction is not due to a difference in conformational motility between form I and form II molecules, but is due to unpaired or weakly hydrogen bonded, localized region(s) of the form I allomorph of circular DNA.