Full-matrix least-squares refinement of lysozymes and analysis of anisotropic thermal motion

Crystal structures of turkey egg lysozyme (TEL) and human lysozyme (HL) were refined by full-matrix least-squares method using anisotropic temperature factors. The refinement converged at the conventional R-values of 0.104 (TEL) and 0.115 (HL) for reflections with F_o > 0 to the resolution of 1.12 Å and 1.15 Å, respectively. The estimated r.m.s. coordinate errors for protein atoms were 0.031 Å (TEL) and 0.034 Å (HL). The introduction of anisotropic temperature factors markedly reduced the R-value but did not significantly affect the main chain coordinates. The degree of anisotropy of atomic thermal motion has strong positive correlation with the square of distance from the molecular centroid. The ratio of the radial component of thermal ellipsoid to the r.m.s. magnitude of three principal components has negative correlation with the distance from the molecular centroid, suggesting the domination of libration rather than breathing motion. The TLS model was applied to elucidate the characteristics of the rigid-body motion. The TLS tensors were determined by the least-squares fit to observed temperature factors. The profile of the magnitude of reproduced temperature factors by the TLS method well fitted to that of observed B_eqv. However, considerable disagreement was observed in the shape and orientation of thermal ellipsoid for atoms with large temperature factors, indicating the large contribution of local motion. The upper estimate of the external motion, 67% (TEL) and 61% (HL) of B_eqv, was deduced from the plot of the magnitude of TLS tensors determined for main chain atoms which were grouped into shells according to the distance from the center of libration. In the external motion, the translational portion is predominant and the contribution of libration and screw motion is relatively small. The internal motion, estimated by subtracting the upper estimate of the external motion from the observed temperature factor, is very similar between TEL and HL in spite of the difference in 54 of 130 amino acid residues and in crystal packing, being suggested to reflect the intrinsic internal motion of chicken-type lysozymes. Proteins 30:232–243, 1998. © 1998 Wiley-Liss, Inc.     

Crystallographic evaluation of internal motion of human alpha-lactalbumin refined by full-matrix least-squares method

The low temperature form of human alpha-lactalbumin (HAL) was crystallized from a 2H2O solution and its structure was refined to the R value of 0.119 at 1.15 A resolution by the full-matrix least-squares method. Average estimated standard deviations of atomic parameters for non-hydrogen atoms were 0.038 A for coordinates and 0.044 A2 for anisotropic temperature factors (Uij). The magnitude of equivalent isotropic temperature factors (Ueqv) was highly correlated with the distance from the molecular centroid and fitted to a quadratic equation as a function of atomic coordinates. The atomic thermal motion was rather isotropic in the core region and the anisotropy increased towards the molecular surface. The statistical analysis revealed the out-of-plane motion of main-chain oxygen atoms, indicating that peptide groups are in rotational vibration around a Calpha.Calpha axis. The TLS model, which describes the rigid-body motion in terms of translation, libration, and screw motions, was adopted for the evaluation of the molecular motion and the TLS parameters were determined by the least-squares fit to Uij. The reproduced Ueqvcal from the TLS parameters was in fair agreement with observed Ueqv, but differences were found in regions of residues, 5-22, 44-48, 70-75, and 121-123, where Ueqv was larger than Ueqvcal because of large local motions. To evaluate the internal motion of HAL, the contribution of the rigid-body motion was determined to be 42.4 % of Ueqv in magnitude, which was the highest estimation to satisfy the condition that the Uijint tensors of the internal motion have positive eigen values. The internal motion represented with atomic thermal ellipsoids clearly showed local motions different from those observed in chicken-type lysozymes which have a backbone structure very similar to HAL. The result indicates that the internal motion is closely related to biological function of proteins.     

Atomic resolution structure of prokaryotic phospholipase A2: analysis of internal motion and implication for a catalytic mechanism

We have found a secreted phospholipase A(2) (PLA(2), EC 3.1.1.4) from Streptomyces violaceoruber A-2688, which is the first PLA(2) identified in prokaryote, and determined its tertiary structure by NMR and X-ray analyses. In this study, we collected the X-ray diffraction data of the bacterial PLA(2) at room temperature (297 K) using conventional MoK(alpha) radiation and refined the structure at a 1.05 A resolution. The atomic resolution analysis led us to introduce disordered conformations and hydrogen atoms into a full anisotropic model. The molecular motion, which is expressed as the sum of rigid-body motion and internal motion of protein, is roughly estimated as the thermal motion when the X-ray diffraction data are collected at room temperature. In this study, we applied a TLS (rigid-body motion in terms of translation, libration, and screw motions) model to analyze the rigid-body motion of the bacterial PLA(2) and calculated the internal motion by subtracting the estimate of the rigid-body motion from the observed anisotropic temperature factor. We also subjected the TLS model to estimate the internal motion of the bovine pancreatic PLA(2) using the anisotropic temperature factor deposited in the Protein Data Bank. Both results indicate that the localization of regions exhibiting larger internal motion in the bacterial PLA(2) is almost the same as that in the bovine pancreatic PLA(2), suggesting that although the tertiary structure of the bacterial PLA(2) is strikingly different from that of the bovine pancreatic PLA(2), the internal motion, which is associated with the calcium(II) ion-binding, phospholipid-binding, and allosteric interfacial activation, is commonly observed in both PLA(2)s.     

Crystallographic analysis of the thermal motion of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with hexamethylenetetramine

The crystal structure of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with hexamethylenetetramine was determined at temperatures of 123, 173, 223, and 293 K. The rigid-body motion of the host and guest molecules was evaluated by means of the TLS method that represents the molecular motion in terms of translation, libration, and screw motion. In increasing the temperature from 123 to 293 K, the amplitude of the rigid body vibration of the host molecule was increased from 1.0 to 1.3 degrees in the rotational motion and from 0.16 to 0.17 A in the translational motion. The cyclomaltoheptaose molecule has the flexibility in seven alpha-(1-->4)-linkages, and each glucose unit was in the rotational vibration around an axis through two glycosidic oxygen atoms. As a result, the rigid-body parameters of cyclomaltoheptaose were considered to be overestimated because of including the contribution from the local motion of glucose units. In contrast, for the guest molecule having no structural flexibility, the TLS analysis demonstrated that the atomic thermal vibration was mostly derived from the rigid body motion. The rotational amplitude of hexamethylenetetramine was changed from 5.2 to 6.6 degrees in increasing the temperature from 123 to 293 K, while the change of the translational amplitude was from 0.20 to 0.23 A. The translational motion of the guest molecule was hindered by the inside wall of the host cavity. The molecular motion was characterized by the rotational vibration around the axis through two nitrogen atoms that were involved in the hydrogen-bond formation.     

An atomic resolution structure for human fibroblast growth factor 1

A 1.10-A atomic resolution X-ray structure of human fibroblast growth factor 1 (FGF-1), a member of the beta-trefoil superfold, has been determined. The beta-trefoil is one of 10 fundamental protein superfolds and is the only superfold to exhibit 3-fold structural symmetry (comprising 3 "trefoil" units). The quality of the diffraction data permits unambiguous assignment of Asn, Gln, and His rotamers, Pro ring pucker, as well as refinement of atomic anisotropic displacement parameters (ADPs). The FGF-1 structure exhibits numerous core-packing defects, detectable using a 1.0-A probe radius. In addition to contributing to the relatively low thermal stability of FGF-1, these defects may also permit domain motions within the structure. The availability of refined ADPs allows a translation/libration/screw (TLS) analysis of putative rigid body domains. The TLS analysis shows that beta-strands 6-12 together form a rigid body, and there is a clear demarcation in TLS motions between the adjacent carboxyl- and amino-termini. Although separate from beta-strands 6-12, the individual beta-strands 1-5 do not exhibit correlated motions; thus, this region appears to be comparatively flexible. The heparin-binding contacts of FGF-1 are located within beta-strands 6-12; conversely, a significant portion of the receptor-binding contacts are located within beta-strands 1-5. Thus, the observed rigid body motion in FGF-1 appears related to the ligand-binding functionalities.     

Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: evidence for an uncoupled R state.

In the R form of ATCase complexed with the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, large temperature factors are reported for the allosteric domains of the regulatory chains. We studied the conformational flexibility of the holoenzyme with molecular dynamics simulations and rigid body (TLS) analysis. The results of the molecular dynamics simulations suggest that, although local atomic fluctuations account for the temperature factors of the catalytic and zinc domains, they do not account for the large temperature factors of the allosteric regions. However, the temperature factors of the allosteric domains can be satisfactorily analyzed using a rigid body model. The simulations and rigid body analysis support the idea that the allosteric regions are mechanically uncoupled from the rest of the enzyme in the PALA structure. Implications of this uncoupling for allosteric regulation are discussed.     

Dynamic transition associated with the thermal denaturation of a small Beta protein

We studied the temperature dependence of the picosecond internal dynamics of an all-beta protein, neocarzinostatin, by incoherent quasielastic neutron scattering. Measurements were made between 20 degrees C and 71 degrees C in heavy water solution. At 20 degrees C, only 33% of the nonexchanged hydrogen atoms show detectable dynamics, a number very close to the fraction of protons involved in the side chains of random coil structures, therefore suggesting a rigid structure in which the only detectable diffusive movements are those involving the side chains of random coil structures. At 61.8 degrees C, although the protein structure is still native, slight dynamic changes are detected that could reflect enhanced backbone and beta-sheet side-chain motions at this higher temperature. Conversely, all internal dynamics parameters (amplitude of diffusive motions, fraction of immobile scatterers, mean-squared vibration amplitude) rapidly change during heat-induced unfolding, indicating a major loss of rigidity of the beta-sandwich structure. The number of protons with diffusive motion increases markedly, whereas the volume occupied by the diffusive motion of protons is reduced. At the half-transition temperature (T = 71 degrees C) most of backbone and beta-sheet side-chain hydrogen atoms are involved in picosecond dynamics.     

Domain motions of glucosamine-6P synthase: comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation

Glucosamine-6-phosphate synthase channels ammonia over 18 A from glutamine at the glutaminase site to fructose-6P at the synthase site. We have modeled the anisotropic displacements of the glutaminase and synthase domains from the two crystallized states, the enzyme in complex with fructose-6P or in complex with glucose-6P and a glutamine affinity analog, using TLS (rigid-body motion in terms of translation, libration, and screw motions) refinement implemented in REFMAC. The domains displacements in the crystal lattices are compared to the movement of the glutaminase domain relative to the synthase domain that occurs during the catalytic cycle upon glutamine binding, which was visualized by comparing the two structures. This movement was analyzed by the program DYNDOM as a 22.8 degrees rotation around an effective hinge axis running approximately parallel to helix 300-317 of the synthase domain, the glutaminase loop that covers the glutaminase site upon glutamine binding acting as the mechanical hinge.     

Anisotropic dynamics of the JE-2147-HIV protease complex: drug resistance and thermodynamic binding mode examined in a 1.09 A structure

The structure of HIV protease (HIV Pr) bound to JE-2147 (also named AG1776 or KNI-764) is determined here to 1.09 A resolution. This highest-resolution structure for HIV Pr allows refinement of anisotropic displacement parameters (ADPs) for all atoms. Clustering based on the directional information in ADPs defines two sets of subdomains such that within each set, subdomains undergo similar anisotropic motion. These sets are (a) the core of monomer A grouped with both substrate-binding flaps and (b) the core of monomer B coupled to both catalytic aspartates (25A/B). The four-stranded beta-sheet (1-4 A/B and 95-99 A/B) that forms a significant part of the dimer interface exhibits large anisotropic amplitudes that differ from those of the other sets of subdomains. JE-2147 is shown here to be a picomolar inhibitor (K(i) = 41 +/- 18 pM). The structure is used to interpret the mechanism of association of JE-2147, a second-generation inhibitor for which binding is enthalpically driven, with respect to first-generation inhibitors for which binding is predominantly entropically driven [Velazquez-Campoy, A., et al. (2001) Arch. Biochem. Biophys. 390, 169-175]. Relative to the entropically driven inhibitor complexes, the JE-2147-HIV Pr complex exhibits an approximately 0.5 A movement of the substrate flaps in toward the substrate, suggesting a more compatible enthalpically driven association. Domains of the protease identified by clustering of ADPs also suggest a model of enthalpy-entropy compensation for all HIV Pr inhibitors in which dynamic coupling of the flaps is offset by an increased level of motion of the beta-sheet domain of the dimer interface (1-4 A/B and 95-99 A/B).     

Secondary structure and oligomerization behavior of equilibrium unfolding intermediates of the lambda cro repressor

The thermal unfolding of the wild-type Cro repressor, its disulfide-bridged mutant Cro-V55C (with the Val-55 --> Cys single amino acid substitution), and a CNBr-fragment (13-66)2 of Cro-V55C was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The combined approach reveals that thermal denaturation of Cro-WT and Cro-V55C proceeds in two steps through equilibrium unfolding intermediates. The first thermal transition of the Cro-V55C dimer involves the melting of the alpha-helices and the short beta-strand localized in the N-terminal part of the molecule. This event is accompanied by the formation of tetramers, and also impacts on the hydrogen-bonding interactions of the C-terminal beta-strands. The beta-sheet formed by the C-terminal parts of each polypeptide chain is the major structural feature of the intermediate state of Cro-V55C and unfolds during a second thermal transition, which is accompanied by the dissociation of the tetramers. Cutting of 12 amino acids in the N-terminal region is sufficient to prevent the formation of alpha-helical structure in the CNBr-fragment of Cro-V55C, and to induce tetramerization already at room temperature. The tetramers may persist over a broad temperature range, and start to dissociate only upon thermal unfolding of the beta-sheet structure formed by the C-terminal regions. The wild-type protein is a dimer at room temperature and at protein concentrations of 1.8-5.8 mg/mL. At lower concentrations, the dimers are stable until the onset of thermal unfolding, which is accompanied by the dissociation of the dimers into monomers. At higher protein concentrations, the unfolding is more complex and involves the formation of tetramers at intermediate temperatures. At these intermediate temperatures, the Cro-WT has lost all of its alpha-helical structure and also most of its native beta-sheet structure. Upon further temperature increase, a tendency for an intermolecular association of the beta-strands is observed, which may result in irreversible beta-aggregation at high protein concentrations.     

The solution structure of concanavalin A probed by FT-IR spectroscopy

The secondary structural properties of various forms of concanavalin A in solution were investigated by Fourier-transform infrared spectroscopy in the Amide I region. As in the crystal, the solution structure of the native protein consists mainly of antiparallel beta-sheet. Carbohydrate binding does not produce major changes in the overall secondary structure of concanavalin A, but affects infrared bands due to loops and beta-turns. Upon demetallization, the spectrum of concanavalin A shows only a small change in the Amide I band, indicating that whereas the beta-sheet structure is conserved, the tertiary properties may be altered. There are also changes in the bands from the tyrosine residues which are compatible with local changes in structure. Confirming tertiary structural differences, the cation-depleted apoprotein is much less stable, denaturing around 63 degrees C, while the native protein denatures only at temperatures around 85 degrees C. Tetramerization proceeds without significant secondary structural change. However, aggregation of the tetramers leads to a significant decrease of the bands corresponding to beta-sheet structure, and changes in the tyrosine bands.     

Crystal structures of Urtica dioica agglutinin and its complex with tri-N-acetylchitotriose

Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of each domain is maintained by four disulfide bridges. The sequence similarity of the two domains is not high (42 %) but their backbone structures are well superimposed except some loop regions. The chitin binding sites are located on the molecular surface at both ends of the dumbbell-shape molecule. The crystal of the NAG3 complex contains two independent molecules forming a protein-sugar 2:2 complex. One NAG3 molecule is sandwiched between two independent UDA-VI molecules and the other sugar molecule is also sandwiched by one UDA-VI molecule and symmetry-related another one. The sugar binding site of N-terminal domain consists of three subsites accommodating NAG3 while two NAG residues are bound to the C-terminal domain. In each sugar-binding site, three aromatic amino acid residues and one serine residue participate to the NAG3 binding. The sugar rings bound to two subsites are stacked to the side-chain groups of tryptophan or histidine and a tyrosine residue is in face-to-face contact with an acetylamino group, to which the hydroxyl group of a serine residue is hydrogen-bonded. The third subsite of the N-terminal domain binds a NAG moiety with hydrogen bonds. The results suggest that the triad of aromatic amino acid residues is intrinsic in sugar binding of hevein-like domains.     

Structural origin of weakly ordered nitroxide motion in spin‐labeled proteins

A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of β-sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites (131 and 151) and a loop site (82) in T4 lysozyme have been determined. Structures of 131R1 and 151R1 determined at cryogenic or ambient temperature reveal an intraresidue C_α—H···S_δ interaction that immobilizes the disulfide group, consistent with a model in which the internal motions of R1 are dominated by rotations about the two terminal bonds (Columbus, Kálai, Jeko, Hideg, and Hubbell, Biochemistry 2001;40:3828–3846). Remarkably, the 131R1 side chain populates two rotamers equally, but the EPR spectrum reflects a single dominant dynamic population, showing that the two rotamers have similar internal motion determined by the common disulfide-backbone interaction. The anisotropic motion for loop residue 82R1 is also accounted for by a common disulfide-backbone interaction, showing that the interaction does not require a specific secondary structure. If the above observations prove to be general, then significant variations in order and rate for R1 at noninteracting solvent-exposed helical and loop sites can be assigned to backbone motion because the internal motion is essentially constant.     

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

Mapping the core of the beta(2)-microglobulin amyloid fibril by H/D exchange

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.     

Normal mode refinement: crystallographic refinement of protein dynamic structure. I. Theory and test by simulated diffraction data.

A dynamic structure refinement method for X-ray crystallography, referred to as the normal mode refinement, is proposed. The Debye-Waller factor is expanded in terms of the low-frequency normal modes whose amplitudes and eigenvectors are experimentally optimized in the process of the crystallographic refinement. In this model, the atomic fluctuations are treated as anisotropic and concerted. The normal modes of the external motion (TLS model) are also introduced to cover the factors other than the internal fluctuations, such as the lattice disorder and diffusion. A program for the normal mode refinement (NM-REF) has been developed. The method has first been tested against simulated diffraction data for human lysozyme calculated by a Monte Carlo simulation. Applications of the method have demonstrated that the normal mode refinement has: (1) improved the fitting to the diffraction data, even with fewer adjustable parameters; (2) distinguished internal fluctuations from external ones; (3) determined anisotropic thermal factors; and (4) identified concerted fluctuations in the protein molecule.     

Sequential events in ribonuclease A thermal unfolding characterized by two-dimensional infrared correlation spectroscopy

The conformational changes in the thermal denaturation of bovine pancreatic ribonuclease A was followed with infrared spectra and analyzed by second derivative and two-dimensional correlation techniques. By analyzing the sequential events in each transition stage, the results were consistent with a step-wise thermal denaturation mechanism in which the structural adjustment of the N-terminal and the opening of the central structure of the protein come before the main unfolding process. Non-native turns were found to form along with the unfolding of the native structures. The central region that is composed of some beta-sheet and alpha-helical structures was found to be the most stable part that might form the residual structure at high temperatures.     

Evaluation of different culture conditions for high-level soluble expression of human cyclin A2 with pET vector in BL21 (DE3) and spectroscopic characterization of its inclusion body structure

In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A(2) in Escherichia coli BL21(DE3), and demonstrated that the highest protein yield was obtained using TB(no glycerol)+0.5% glucose medium at 25 degrees C. By single immobilized metal ion affinity chromatography, we got highly purified human cyclin A(2) with a yield ranged from 20 to 30 mg/L. By amyloid-diagnostic dye ThT binding and Fourier transform infrared spectroscopy, we observed a significant decrease in alpha-helix content and an increase in beta-sheet structure in cyclin A(2) inclusion body in comparison to its native protein, and confirmed the resemblance of the internal organization of cyclin A(2) inclusion body and amyloid fibrils.