Persistent conformational heterogeneity of triosephosphate isomerase: separation and characterization of conformational isomers in solution

Subunit dissociation of dimeric rabbit muscle triosephosphate isomerase (TIM) by hydrostatic pressure has previously been shown not to follow the expected dependence on protein concentration [Rietveld and Ferreira (1996) Biochemistry 35, 7743-7751]. This anomalous behavior was attributed to persistent conformational heterogeneity (i.e., the coexistence of long-lived conformational isomers) in the ensemble of TIM dimers. Here, we initially show that subunit dissociation/unfolding of TIM by guanidine hydrochloride (GdnHCl) also exhibits an anomalous dependence on protein concentration. Dissociation/unfolding of TIM by GdnHCl was investigated by intrinsic fluorescence and circular dichroism spectroscopies and was found to be a highly cooperative transition in which the tertiary and secondary structures of the protein were concomitantly lost. A procedure based on size-exclusion chromatography in the presence of intermediate (0.6 M) GdnHCl concentrations was developed to isolate two conformational isomers of TIM that exhibit significantly different stabilities and kinetics of unfolding by GdnHCl. Complete unfolding of the two isolated conformers at a high GdnHCl concentration (1.5 M), followed by refolding by removal of the denaturant, completely abolished the differences in their unfolding kinetics. These results indicate that such differences stem from conformational heterogeneity of TIM and are not related to any chemical modification of the protein. Furthermore, they add support to the notion that long-lived conformational isomers of TIM coexist in solution and provide a basis for the interpretation of the persistent heterogeneity of this protein.     

Human neuromodulator SLURP-1: Bacterial expression, binding to muscle-type nicotinic acetylcholine receptor, secondary structure, and conformational heterogeneity in solution

Human protein SLURP-1 is an endogenous neuromodulator belonging to the Ly-6/uPAR family and acting on nicotinic acetylcholine receptors. In the present work, the gene of SLURP-1 was expressed in E. coli. The bacterial systems engineered for SLURP-1 expression as fused with thioredoxin and secretion with leader peptide STII failed in the production of milligram quantities of the protein. The SLURP-1 was produced with high-yield in the form of inclusion bodies, and different methods of the protein refolding were tested. Milligram quantities of recombinant SLURP-1 and its ¹⁵N-labeled analog were obtained. The recombinant SLURP-1 competed with ¹²⁵I-α-bungarotoxin for binding to muscle-type Torpedo californica nAChR at micromolar concentrations, indicating a partial overlap in the binding sites for SLURP-1 and α-neurotoxins on the receptor surface. NMR study revealed conformational heterogeneity of SLURP-1 in aqueous solution, which was associated with cis-trans isomerization of the Tyr39-Pro40 peptide bond. The two structural forms of the protein have almost equal population in aqueous solution, and exchange process between them takes place with characteristic time of about 4 ms. Almost complete ¹H and ¹⁵N resonance assignment was obtained for both structural forms of SLURP-1. The secondary structure of SLURP-1 involves two antiparallel β-sheets formed from five β-strands and closely resembles those of three-finger snake neurotoxins.     

On the structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution.

The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.     

Protein conformational dynamics of crambin in crystal, solution and in the trajectories of molecular dynamics simulations

Atomic displacement parameters — B factors of the eight crambin crystal structures obtained at 0.54–1.5 Å resolution and temperatures of 100–293 K have been analyzed. The comparable contributions to the B factor values are the intramolecular motions which are modeled by the harmonic vibration calculations and derived from the molecular dynamics simulation (MD) as well as rigid body changes in the position of a protein molecule as a whole. In solution for the average NMR structure of crambin the amplitudes of the backbone atomic fluctuations of the most residues of the segments with the regular backbone conformations are close to the amplitudes of the small scale harmonic vibrations. For the same residues the probability of the medium scale fluctuations fixed by the hydrogen exchange method is very low. The restricted conformational mobility of those segments is coupled with the depressed amplitudes of the fluctuation changes of the tertiary structure registered by the residue accessibility changes in an ensemble of NMR structures that forms the average NMR structure of crambin. The amplitudes of temperature fluctuations of backbone atoms and the tertiary structure raise in the segment with the irregular conformations, turn and loops. In the same segments the amplitudes of the calculated harmonic vibrations also increase, but to a lesser extent and especially in the interhelical loop with the most strong and complicated fluctuation changes of the backbone conformation. In solution for the NMR structure in this loop the conformational transitions occur between the conformational substates separated by the energy barriers, but they are not observed even in the long 100 ns trajectories from the MD simulation of crambin. These strong local fluctuation changes of the structure may play a key role in the protein functioning and modern performance improvements in the MD simulation techniques are oriented to increase the probability of protein appearance in the trajectories from the MD simulations.     

Spatial heterogeneity, social structure and disease dynamics of animal populations

Social groupings, population dynamics and population movements of animals all give rise to spatio-temporal variations in population levels. These variations may be of crucial importance when considering the spread of infectious diseases since infection levels do not increase unless there is a sufficient pool of susceptible individuals. This paper explores the impact of social groupings on the potential for an endemic disease to develop in a spatially explicit model system. Analysis of the model demonstrates that the explicit inclusion of space allows asymmetry between groups to arise when this was not possible in the equivalent spatially homogeneous system. Moreover, differences in movement behaviours for susceptible and infected individuals gives rise to different spatial profiles for the populations. These profiles were not observed in previous work on an epidemic system. The results are discussed in an ecological context with reference to furious and dumb strains of infectious diseases.     

The dynamics,structure, and conformational free energy of proline-containing antifreeze glycoprotein

Recent NMR studies of the solution structure of the 14-amino acid antifreeze glycoprotein AFGP-8 have concluded that the molecule lacks long-range order. The implication that an apparently unstructured molecule can still have a very precise function as a freezing inhibitor seems startling at first consideration. To gain insight into the nature of conformations and motions in AFGP-8, we have undertaken molecular dynamics simulations augmented with free energy calculations using a continuum solvation model. Starting from 10 different NMR structures, 20 ns of dynamics of AFGP were explored. The dynamics show that AFGP structure is composed of four segments, joined by very flexible pivots positioned at alanine 5, 8, and 11. The dynamics also show that the presence of prolines in this small AFGP structure facilitates the adoption of the poly-proline II structure as its overall conformation, although AFGP does adopt other conformations during the course of dynamics as well. The free energies calculated using a continuum solvation model show that the lowest free energy conformations, while being energetically equal, are drastically different in conformations. In other words, this AFGP molecule has many structurally distinct and energetically equal minima in its energy landscape. In addition, conformational, energetic, and hydrogen bond analyses suggest that the intramolecular hydrogen bonds between the N-acetyl group and the protein backbone are an important integral part of the overall stability of the AFGP molecule. The relevance of these findings to the mechanism of freezing inhibition is discussed.     

Crystal structure,conformational analysis,and molecular dynamics of tetra-O-methyl-(+) -catechin

The structure of tetra-O-methyl- (+) -catechin has been determined in the crystalline state. Two independent molecules, denoted structure A and structure B, exist in the unit cell. Crystals are triclinic, space group P1, a = 4.8125(2) Å, b = 12.9148(8) Å, c = 13.8862(11) Å, α = 86.962(6) °, β = 89.120(5)°, γ = 88.044(5)°, Z = 2, D_c = 1.336 g cm^?3, R = 0.033 for 6830 observations. The heterocyclic rings of the crystal structures are compared to previous results for 8-bromotetra-O-methyl-(+)-catechin, penta-O-acetyl-(+)-catechin, and (?) -epicatechin. One of the two molecules has a heterocyclic ring conformation similar to that observed previously for (?)-epicatechin, and the other has a heterocyclic ring conformation similar to one predicted earlier in a theoretical analysis of dimers of (+)-catechin and (?) -epicatechin. Both structure A and structure B in the crystal have heterocyclic ring conformations that place the dimethoxyphenyl substituent at C(2) in the equatorial position. However, this heterocyclic ring conformation does not explain the proton nmr coupling constant measured in solution. Molecular dynamics simulations show an equatorial ? axial interconversion of the heterocyclic ring, which can explain the nmr results. © 1993 John Wiley & Sons, Inc.     

3nion-independent conformational ordering in iota-carrageenan: Disorder-order equilibria and dynamics

The equilibria and dynamics of the disorder-to-order transition of the anionic polysaccharide iota-carrageenan have been studied in the presence of tetramethyl-ammonium salts. By the use of a stopped-flow polarimeter, the rate equation and temperature dependence of the observed forward rate-constant were found to accord with a co-operative dimerisation process. Activation parameters for helix nucleation were shown to be independent of the anion for solutions containing tetramethylammonium chloride and bromide, i.e., ΔH^‡ = 1 ±3 kJ.mol⁻¹, ΔS^‡ = −178 ±10 J.mol⁻¹.K⁻¹, ΔG^‡_298K = 54 ±2 kJ.mol⁻¹, and k_nuc,298K = 1880 ±80 dm³.mol⁻¹.s⁻¹. The temperature dependence of optical rotation was also shown to be independent of the anion present.     

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.     

Molecular dynamics simulation in vacuo and in solution of cyclolinopeptide A: a conformational study.

The conformation of cyclolinopeptide A [c-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring peptide with remarkable cytoprotective activity, has been investigated by means of molecular dynamics simulations in various molecular environments. Structural and dynamical properties have been analyzed and compared with those experimentally determined. A detailed analysis of hydrogen bonds is reported.     

Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

Acylphosphatase is a small enzyme that catalyzes the hydrolysis of acyl phosphates. Here, we present the solution structure of acylphosphatase from Bacillus subtilis (BsAcP), the first from a Gram-positive bacterium. We found that its active site is disordered, whereas it converted to an ordered state upon ligand binding. The structure of BsAcP is sensitive to pH and it has multiple conformations in equilibrium at acidic pH (pH < 5.8). Only one main conformation could bind ligand, and the relative population of these states is modulated by ligand concentration. This study provides direct evidence for the role of ligand in conformational selection.     

NMR solution structure and dynamics of motilin in isotropic phospholipid bicellar solution

The structure and dynamics of the gastrointestinal peptide hormone motilin, consisting of 22 amino acid residues, have been studied in the presence of isotropic q=0.5 phospholipid bicelles. The NMR solution structure of the peptide in acidic bicelle solution was determined from 203 NOE-derived distance constraints and six backbone torsion angle constraints. Dynamic properties for the ¹³C-¹H vector in Leu10 were determined for motilin specifically labeled with ¹³C at this position by analysis of multiple-field relaxation data. The structure reveals an ordered -helical conformation between Glu9 and Lys20. The N-terminus is also well structured with a turn resembling that of a classical -turn. The ¹³C dynamics clearly show that motilin tumbles slowly in solution, with a correlation time characteristic of a large object. It was also found that motilin has a large degree of local flexibility as compared with what has previously been reported in SDS micelles. The results show that motilin interacts with the bicelle, displaying motional properties of a peptide bound to a membrane. In comparison, motilin in neutral bicelles seems less structured and more flexible. This study shows that the small isotropic bicelles are well suited for use as membrane-mimetic for structural as well as dynamical investigations of membrane-bound peptides by high-resolution NMR.     

The solution structure and dynamics of an Arc repressor mutant reveal premelting conformational changes related to DNA binding.

The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor. A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region. For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found. In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer. While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, where the Phe 10 ring contacts the phosphate backbone [Raumann, B. E., et al. (1994) Nature 367, 754-757]. As well as DNA binding, denaturation by urea and high temperatures induces the functionally active "out" conformation. With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor. The dynamical properties of the Arc-MYL and the wild-type Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments. The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding.     

Torsion angle dynamics: Reduced variable conformational sampling enhances crystallographic structure refinement

A reduced variable conformational sampling strategy for macromolecules based on molecular dynamics in torsion angle space is evaluated using crystallographic refinement as a prototypical search problem. Bae and Haug's algorithm for constrained dynamics [Bae, D. S., Haug, E. J. A recursive formulation for constrained mechanical system dynamics. Mech. Struct. Mach. 15:359–382, 1987], originally developed for robotics, was used. Their formulation solves the equations of motion exactly for arbitrary holonomic constraints, and hence differs from commonly used approximation algorithms. It uses gradients calculated in Cartesian coordinates, and thus also differs from internal coordinate formulations. Molecular dynamics can be carried out at significantly higher temperatures due to the elimination of the high frequency bond and angle vibrations. The sampling strategy presented here combines high temperature torsion angle dynamics with repeated trajectories using different initial velocities. The best solutions can be identified by the free R value, or the R value if experimental phase information is appropriately included in the refinement. Applications to crystallographic refinement show a significantly increased radius of convergence over conventional techniques. For a test system with diffraction data to 2 Å resolution, slow-cooling protocols fail to converge if the backbone atom root mean square (rms) coordinate deviation from the crystal structure is greater than 1.25 Å, but torsion angle refinement can correct backbone atom rms coordinate deviations up to approximately 1.7 Å. © 1994 Wiley-Liss, Inc.     

NMR solution structure and backbone dynamics of the CC chemokine eotaxin-3.

Eotaxin-3 is one of three related chemokines that specifically activate chemokine receptor CCR3. We report the 3D structure and backbone dynamics of eotaxin-3 determined by NMR spectroscopy. Eotaxin-3 is monomeric under the conditions in this study and consists of an unstructured N-terminus before the first two conserved cysteine residues, an irregularly structured N-loop following the second conserved cysteine, a single turn of 3(10)-helix, a three-stranded antiparallel beta-sheet, an alpha-helix, and an unstructured C-terminal tail. As in other chemokines, the alpha-helix packs against one face of the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 9-65) are 0.44 and 1.01 A, respectively. A comparison between the structures of eotaxin-3 and related chemokines suggests that the electrostatic potential in the vicinity of a surface groove and the structure of the beta2-beta3 turn may be important for maintaining receptor specificity. The backbone dynamics of eotaxin-3 were determined from 15N NMR relaxation data using the extended model free dynamics formalism. Large amplitude motions on the picosecond to nanosecond time scale were observed in both termini and in some residues in the N-loop, the beta1-beta2 turn, and the beta3 strand; the location of these residues suggests a possible role for dynamics in receptor binding and activation. In contrast to eotaxin, eotaxin-3 exhibits no substantial mobility on the microsecond to millisecond time scale.     

The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5

The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand-specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine-dependent and factor-independent activation of the receptor.