Conformational changes, dynamics and assignments in 1H NMR studies of proteins using ring current calculations. Hen egg white lysozyme.
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Acetylglucosamine↗
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Effect of aspirin on conformation and dynamics of membrane proteins in platelets and erythrocytes.
The effect of the chemical modifications induced by aspirin (acetylsalicylic acid), acetyl chloride or salicylate on platelet membranes and erythrocyte ghosts has been investigated by means of fluorescence quenching and ESR spectroscopy in relation to our earlier findings of acetylation-induced reduction of platelet and erythrocyte membrane lipid fluidity. Only aspirin was found to induce disorders in the lipid-protein matrix and membrane protein conformation. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased after aspirin action in both platelet and erythrocyte membranes. This resulted in a significant increase in the maximum energy transfer efficiency. The decrease in the ratio of the amplitudes of low-field peaks of weakly to strongly immobilized fractions of maleimide spin label (4-maleimido-2,2,6-6-tetramethylpiperidine-1-oxyl) and the rise in the relative rotational correlation time of iodoacetamide spin label [4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl] indicate that aspirin effectively immobilizes membrane proteins in the plane of the lipid bilayer, whereas neither acetyl chloride or salicylate gave rise to detectable effects. We conclude that aspirin-induced alterations in membrane protein structure induce a reorganization of lipid assembly as well as rearrangements in the membrane protein pattern, and consequently alterations in lipid-protein interactions. Thus, the interaction of aspirin with platelet and erythrocyte membranes may induce local conformational changes in membranes, which are discussed in connection with impairment of platelet function. A new mode of protein chemical modification by aspirin is suggested which involves the generation of reactive salicylic residue during the fast degradation of aspirin under physiological conditions.
Anilino Naphthalenesulfonates↗
NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases.
Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.
Cloning, Molecular↗
Conformational and dynamic considerations in the design of peptide hormone analogs.
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Amino Acid Sequence↗
Calculations of protein circular dichroism from first principles.
Understanding the relationship between the amino acid sequence of a protein and its unique, compact 3D structure is one of the grand challenges in molecular biophysics. One particularly exciting approach is time-resolved electronic circular dichroism (CD) spectroscopy, which offers resolution on a nanosecond (or faster) time scale, although it does not provide the spatial resolution of techniques like X-ray crystallography or NMR. The thrust of our work is to underpin fast time scale spectroscopic studies of protein folding with a stronger theoretical foundation. Ultimately, we seek to use molecular dynamics simulations to study the influence of conformational dynamics and conformational transitions on the electronic CD spectra of proteins. We discuss how improved quantum chemical models of individual chromophores, including aromatic sidechains, can be incorporated into calculations of the electronic structure of proteins and their CD.
Bacterial Proteins↗
Conformation and dynamics of a left-handed Z-DNA hairpin: studies of d(CGCGCGTTTTCGCGCG) in solution.
The physical properties of the DNA oligomer d(CGCGCGTTTTCGCGCG) in solvents containing 4 M NaClO4 and 0.1 M NaCl were investigated by proton NMR, optical melting, and circular dichroism spectroscopy. Results of these investigations are as follows: (i) The DNA hexadecamer exists as a unimolecular hairpin in either high or low salt. (ii) In high salt the stem region of the hairpin is in the left-handed Z conformation. (iii) In either high or low salt, the duplex stem of the hairpin is stabilized against melting by approximately 40 degrees C compared to the linear core duplex. The added stability of the hairpin is entropic in origin. (iv) In high salt, as the temperature is elevated, the equilibrium structure of the duplex stem of the hairpin shifts from the Z to the B conformation before melting. (v) In low salt, when the DNA duplex exists in the B conformation, attachment of a T4 single-strand loop to one end only slightly decreases (by 14%) the correlation time of the CH5-CH6 interproton vector. In high salt, when the DNA duplex exists in the Z conformation, the correlation time of the CH5-CH6 interproton vector decreases by 51%. Since these viscosity-corrected correlation times are taken to be indicators of duplex motions on the nanosecond time scale, this result directly suggests a larger amplitude of these motions is present in the duplex stem of the hairpin when it exists in the Z conformation.
Circular Dichroism↗
Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands.
The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds L-histidine and L-arginine (tightly) and L-lysine and L-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation. Surprisingly, various liganded HisJ forms display substantial differences in their physicochemical characteristics and capacity to induce the ATPase. This is due to either different effects of the individual ligands on the respective closed structures, or to different equilibria being reached for each ligand between the open liganded form and the closed liganded form [Wolf, A. , Lee, K.C., Kirsch, J.F. & Ames, G.F.-L. (1996) J. Biol. Chem. 271, 21243-21250]. In this work, time-resolved measurements of the decay of intrinsic HisJ fluorescence and of the decay of the anisotropy of the fluorescence, as well as the analysis of the steady-state near UV CD and fluorescence spectra, rule out the model in which the differences between liganded complexes reflect different equilibria. The decay of the anisotropy of the fluorescence shows that liganded complexes differ dramatically in their large-scale conformational dynamics. Differential scanning calorimetry (DSC) curves for the HisJ thermal unfolding are well described by a scheme of equilibrium two-state unfolding of two independent domains, which can be ascribed to the two-domain structure of HisJ. This is true both for apo-HisJ at various pH values, and for HisJ in the presence of its ligands at varying concentrations, at pH 8.3. The DSC and structural data suggest that all ligands interact more extensively with the larger domain. A qualitative model for the HisJ conformational dynamics employing the idea of a twisting movement of the domains is proposed, which explains the difference in the efficacy of the ATPase induction by the various liganded HisJ forms.
ATP-Binding Cassette Transporters↗
Effects of trifluoperazine on the conformation and dynamics of membrane proteins in human erythrocytes.
The chemical modifications induced by trifluoperazine (TFP) in erythrocyte ghosts have been investigated by fluorescence quenching. The apparent distance separating the membrane protein tryptophans and bound 1-aniline-8-naphthalene sulfonate (ANS) molecules decreased after treating erythrocyte membranes with TFP. This effect was accompanied by a significant decrease in the maximum efficiency of energy transfer. We conclude that TFP-induced alterations in the structure of membrane proteins lead to a rearrangement of the surrounding lipids, and consequently to local conformational changes in membrane organization.
Anilino Naphthalenesulfonates↗
Actin and temperature effects on the cross-linking of the SH1-SH2 helix in myosin subfragment 1.
Past biochemical work on myosin subfragment 1 (S1) has shown that the bent alpha-helix containing the reactive thiols SH1 (Cys(707)) and SH2 (Cys(697)) changes upon nucleotide and actin binding. In this study, we investigated the conformational dynamics of the SH1-SH2 helix in two actin-bound states of myosin and examined the effect of temperature on this helix, using five cross-linking reagents that are 5-15 A in length. Actin inhibited the cross-linking of SH1 to SH2 on both S1 and S1.MgADP for all of the reagents. Because the rate of SH2 modification was not altered by actin, the inhibition of cross-linking must result from a strong stabilization of the SH1-SH2 helix in the actin-bound states of S1. The dynamics of the helix is also influenced by temperature. At 25 degrees C, the rate constants for cross-linking in S1 alone are low, with values of approximately 0.010 min(-1) for all of the reagents. At 4 degrees C, the rate constants, except for the shortest reagent, range between 0.030 and 0.070 min(-1). The rate constants for SH2 modification in SH1-modified S1 show the opposite trend; they increase with the increases in temperature. The greater cross-linking at the lower temperature indicates destabilization of the SH1-SH2 helix at 4 degrees C. These results are discussed in terms of conformational dynamics of the SH1-SH2 helix.
Actins↗
Clinical gain from improved beam delivery systems.
The feasibility of dynamic conformal heavy charged particle radiotherapy has been investigated at UCLBL, and shows high promise of: 1. an improved therapeutic ratio and 2. reduction in the number of treatment portals required for efficient treatment delivery. Assessment of dose to tumor and critical structures for several anatomical sites have been carried out using a normal tissue complication algorithm developed at LBL. For high-LET charged particle treatment delivery, dynamic conformal therapy using a raster scanned beam with variable modulation and multileaf collimator appears to be the optimal technique for treatment delivery.
Energy Transfer↗
Tryptophanyl substitutions in apomyoglobin affect conformation and dynamic properties of AGH subdomain.
The solvent accessibilities to the tryptophanyl microenvironments of wild type sperm whale apomyoglobin (apoMb) and two mutants (W7F and W14F) containing a single tryptophan are measured by fluorescence quenching studies. The results are compared to those relative to horse apoMb. In the wild type sperm whale protein, no difference is noticed in the solvent accessibility of the two indole residues, as documented by the values of the Stern-Volmer constants. By contrast, the two tryptophan residues of horse apoMb are exposed to the solvent in a different way, thus indicating that some local conformational differences exist between the two homologous proteins in solution. The single W --> F substitution at either position 7 or 14 determines local conformational changes that increase the accessibility of the remaining indole residue but do not affect the overall architecture of the protein molecule.
Acrylamide↗
Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.
MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.
Amino Acid Sequence↗
Elevated temperature and tyrosine iodination aid in the crystallization and structure determination of an antifreeze protein.
Production and refolding of recombinant Choristoneura fumiferana antifreeze protein (CfAFP) leads to a disulfide-bonded product containing dynamic conformational microheterogeneity. Difficulties in the crystallization of this protein arising from its microheterogeneity were overcome by screening of crystallization conditions at various temperatures and finally using a temperature of 318 K to obtain diffraction-quality crystals. In addition, heavy-atom derivatization of this protein required the iodination of a specific tyrosine residue, leading to the successful single anomalous scattering (SAS) structure determination. The techniques of higher temperature screening, to reduce dynamic conformational microheterogeneity, and defined tyrosine iodination, for specific heavy-atom incorporation, are methods which can be employed with other proteins to aid in structure determination.
Animals↗
31P NMR investigation of the backbone conformation and dynamics of the hexamer duplex d(5'-GCATGC)2 in its complex with the antibiotic nogalamycin.
Heteronuclear chemical shift correlation experiments confirm that the two down-field shifted 31P resonances in the spectrum of the (nogalamycin)2-d(GCATGC)2 complex correspond to the phosphodiesters CpA and TpG at the intercalation sites. 31P relaxation measurements (R1, R2 and [1H]-31P NOE) at 4.7 and 9.4 T permit the correlation time of each phosphate to be determined together with their chemical shift anisotropies. Significant differences in deoxyribose H3'-31P coupling constants and chemical shift anisotropy contributions are observed, consistent with an asymmetric DNA backbone conformation for the phosphate groups at the intercalation sites. Large amplitude internal motions of the phosphates do not appear to contribute significantly to relaxation.
DNA↗
Stereotactic radiosurgery for brain AVMs: role of interobserver variation in target definition on digital subtraction angiography.
PURPOSE: We evaluated the extent of interobserver variation in contouring arteriovenous malformations (AVMs) on digital subtraction angiography (DSA) with respect to volume, spatial localization, and dosimetry and correlated our findings with the clinical outcome. METHODS AND MATERIALS: Thirty-one patients who had undergone radiosurgery for brain AVMs were studied. Six clinicians independently contoured the nidus on the original DSA. As a measure of variation, the ratio between the volumes of agreement and the corresponding encompassing volumes, as well as the absolute positional shift between the individual target volumes were derived. Using the original treatment plan, the dosimetric coverage of the individually contoured volumes with standard collimators was compared with a similar plan using dynamic conformal arcs. RESULTS: The mean contoured nidus volume was 3.6 +/- 5.6 cm3. The mean agreement ratio was 0.45 +/- 0.18 for all possible pairs of observers. The mean absolute positional shift between individually contoured volumes was 2.8 +/- 2.6 mm. These differences were more marked in previously treated groups and tended to be more pronounced in those with treatment failure. The mean coverage of the individual volumes by the 80% prescription isodose was 88.1% +/- 3.2% using conventional collimators and 78.9% +/- 4.4% using dynamic conformal arcs (p = 0.001). CONCLUSION: Substantial interobserver variations exist when contouring brain AVMs on DSA for the purpose of radiosurgical planning. Such variations may result in underdosage to the AVM and, thereby, contribute to treatment failure. The consequences of contouring variations may increase with the use of more conformal radiosurgical techniques.
Adolescent↗
Fluorescence of the single tryptophan of cutinase: temperature and pH effect on protein conformation and dynamics.
The cutinase from Fusarium solani pisi is an enzyme with a single L-tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala-Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge-transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10-fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling-in-cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein-melting temperature and to protein-backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.
Animals↗
The conformational and dynamic basis for ligand binding reactivity in hemoglobin Ypsilanti (beta 99 asp-->Tyr): origin of the quaternary enhancement effect.
Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxygen with little or no cooperativity and with high affinity [Doyle, M. L., et al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81-91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new quaternary structure that partially resembles the recently described R2 structure [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very little is known about either the solution phase conformations of the liganded and deoxy forms of HbY or the molecular basis for the large quaternary enhancement effect (Doyle et al., 1992). In this study, near-IR absorption, Soret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are used to probe the liganded and deoxy derivatives of HbY in solution. Nanosecond time-resolved near-IR absorption measurements are used to expose the relaxation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebinding curves for photodissociated COHbY. The spectroscopic results indicate that COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha 1 beta 2 interface. The deoxy derivative of HbY has spectroscopic features that are very similar to those observed for species assigned to the deoxy R or half-liganded R conformations of human adult hemoglobin (HbA). The 10 ns to 100 micros relaxation properties of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observed. The near-absence of relaxation in the HbY photoproduct explains the differences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 1992). In addition, the spectroscopic data and the X-ray crystallographic results explain the slow relaxation for HbY and the near-absence of cooperative ligand binding for this protein based on the behavior of the penultimate tyrosines.
Adult↗
Adsorption of single DNA molecules at the water/fused-silica interface.
We applied total internal reflection fluorescence microscopy (TIRFM) to study intermolecular interactions at the water/fused-silica interface at the single-molecule level. Real-time molecular motion at the interface was recorded to reveal adsorption behavior and conformational dynamics of three DNAs with sticky ends of different numbers of unpaired bases. Features of DNA motion at the interface, such as evanescent-field residence time, linear velocity and frequency of adsorption/desorption events were measured to assess the relative affinities of the oligonucleotides for the surface. The general trend of stronger interaction with the surface for longer sticky ends confirmed hydrophobic interaction and hydrogen bonding as the driving forces of DNA adsorption to fused-silica at pH 5. For DNAs of different sizes, different conformational dynamics and the accessibility of sticky ends give rise to a nonlinear relationship with respect to affinity. Such information may prove valuable for chromatography studies as well as for the design of DNA microarrays and drug delivery systems.
Adsorption↗