Intrinsic nature of the three-dimensional structure of proteins as determined by distance geometry with good sampling properties

Summary A protocol for distance geometry calculation is shown to have excellent sampling properties in the determination of three-dimensional structures of proteins from nuclear magnetic resonance (NMR) data. This protocol uses a simulated annealing optimization employing mass-weighted molecular dynamics in four-dimensional space (Havel, T.F. (1991) Prog. Biophys. Mol. Biol., 56, 43–78). It attains an extremely large radius of convergence, allowing a random coil conformation to be used as the initial estimate for the succeeding optimization process. Computations are performed with four systems of simulated distance data as tests of the protocol, using an unconstrained l-alanine 30mer and three different types of proteins, bovine pancreatic trypsin inhibitor, the -amylase inhibitor Tendamistat, and the N-terminal domain of the 434-repressor. The test of the unconstrained polypeptide confirms that the sampled conformational space is that of the statistical random coil. In the larger and more complicated systems of the three proteins, the protocol gives complete convergence of the optimization without any trace of initial structure dependence. As a result of an exhaustive conformational sampling by the protocol, the intrinsic nature of the structures generated with distance restraints derived from NMR data has been revealed. When the sampled structures are compared with the corresponding X-ray structures, we find that the averages of the sampled structures always show a certain pattern of discrepancy from the X-ray structure. This discrepancy is due to the short distance nature of the distance restraints, and correlates with the characteristic shape of the protein molecule.Abbreviations r.m.s.d. root-mean-square deviation - MD molecular dynamics - NMR nuclear magnetic resonance - NOE nuclear Overhauser enhancement - BPTI bovine pancreatic trypsin inhibitor     

Torsion angle approach to nucleic acid distance geometry: TANDY

Summary An efficient algorithm for generating DNA structures from a given set of distance constraints has been developed. The present implementation is suited for single-stranded DNA. The performance of the program has been tested with constraint sets representative of most stringent theoretical cases and also with usually available experimental ones. The results indicate that use of NOE-derived constraints alone generates an extremely large family of conformers and suggest that the quality of structure determination may be enhanced by incorporating additional constraints obtained by other means. The speed of the program makes it ideal for interactive use in conjunction with other complementary algorithms such as those for spectral simulation, energy minimization and molecular dynamics calculations.Dedicated to the memory of Professor V.F. Bystrov     

Application of a genetic algorithm in the conformational analysis of methylene-acetal-linked thymine dimers in DNA: Comparison with distance geometry calculations

The three-dimensional spatial structure of a methylene-acetal-linked thymine dimer presentin a 10 base-pair (bp) sense–antisense DNA duplex was studied with a geneticalgorithm designed to interpret NOE distance restraints. Trial solutions were represented bytorsion angles. This means that bond angles for the dimer trial structures are kept fixed duringthe genetic algorithm optimization. Bond angle values were extracted from a 10 bpsense–antisense duplex model that was subjected to energy minimization by means ofa modified AMBER force field. A set of 63 proton–proton distance restraints definingthe methylene-acetal-linked thymine dimer was available. The genetic algorithm minimizesthe difference between distances in the trial structures and distance restraints. A largeconformational search space could be covered in the genetic algorithm optimization byallowing a wide range of torsion angles. The genetic algorithm optimization in all cases ledto one family of structures. This family of the methylene-acetal-linked thymine dimer in theduplex differs from the family that was suggested from distance geometry calculations. It isdemonstrated that the bond angle geometry around the methylene-acetal linkage plays animportant role in the optimization.     

A systematic comparison of three structure determination methods from NMR data: Dependence upon quality and quantity of data

Summary We have systematically examined how the quality of NMR protein structures depends on (1) the number of NOE distance constraints. (2) their assumed precision, (3) the method of structure calculation and (4) the size of the protein. The test sets of distance constraints have been derived from the crystal structures of crambin (5 kDa) and staphylococcal nuclease (17 kDa). Three methods of structure calculation have been compared: Distance Geometry (DGEOM), Restrained Molecular Dynamics (XPLOR) and the Double Iterated Kalman Filter (DIKF). All three methods can reproduce the general features of the starting structure under all conditions tested. In many instances the apparent precision of the calculated structure (as measured by the RMS dispersion from the average) is greater than its accuracy (as measured by the RMS deviation of the average structure from the starting crystal structure). The global RMS deviations from the reference structures decrease exponentially as the number of constraints is increased, and after using about 30% of all potential constraints, the crrors asymptotically approach a limiting value. Increasing the assumed precision of the constraints has the same qualitative effect as increasing the number of constraints. For comparable numbers of constraints/residue, the precision of the calculated structure is less for the larger than for the smaller protein, regardless of the method of calculation. The accuracy of the average structure calculated by Restrained Molecular Dynamics is greater than that of structures obtained by purely geometric methods (DGEOM and DIKF).     

Extensive distance geometry calculations with different NOE calibrations: New criteria for structure selection applied to Sandostatin and BPTI

Summary To generate structures efficiently, a version of the distance geometry program DIANA for a parallel computer was developed, new objective criteria for the selection of NMR solution structures are presented, and the influence of using different calibrations of NOE intensities on the final structures are described. The methods are applied to the structure determination of Sandostatin, a disulfide-bridge octapeptide, and to model calculations of BPTI. On an Alliant FX2800 computer using 10 processors in parallel, the calculations were done 9.2 times faster than with a single processor. Up to 7000 Sandostatin structures were calculated with distance and angular constraints. The procedure for selecting acceptable structures is based on the maximum values of pairwise RMSDs between structures. Suitable target function cut-offs are defined independent of the number of starting structures. The method allowed for an objective comparison of three groups of Sandostatin structures that were calculated from different sets of upper distance constraints which were derived from the same NOE intensity data using three empirical calibration curves. The number of converged structures and the target function values differed significantly among the three groups, but the structures were qualitatively and quantitatively very similar. The conformation is well determined in the cyclic region Cys²–Cys⁷ and adopts a -turn centered at d-Trp⁴–Lys⁵. The criteria for structure selection were further tested with BPTI. Results obtained from sets of structures calculated with and without using the REDAC strategy are consistent and suggest that the structure selection method is objective and generally applicable.     

High-resolution structure and dynamic implications for a double-helical gramicidin A conformer

Summary The high-resolution structure of a dimeric conformer of gramicidin A, a 15-residue polypeptide, has been determined in the mixed-solvent system of benzene and ethanol by 2D NMR techniques. NOEs, coupling constants and hydrogen-bond information were used to generate 744 experimental constraints for the dimer. Stereoassignment of most -methylene groups was achieved by analysis of ³J, d(i,i), d_N(i,i) and d_N(i+1,i) distances, and consideration of the initial backbone structure determinations. Stereoassignment of several leucine methyl groups was accomplished via a distance geometry/simulated annealing routine, used for structure determination and refinement. The relatively static backbone structure was determined first and held rigid while side-chain conformations were calculated. This procedure is evaluated versus standard NMR structure determination protocols. The backbone is an antiparallel intertwined double helix, with 5.6–5.7 residues per turn, a total dimer length of 36–37 Å, and a pore width of 2.5–3.0 Å (van der Waals to van der Waals). The structure and dynamics of the side chains are discussed in depth, with careful attention for both the convergence of structures and the residual constraint violations per residue. Side-chain positions impart substantial amphipathic character to the helix, which could influence the conformational change that takes place upon membrane insertion of this channel-forming polypeptide.To whom correspondence should be addressed.     

SESAME: A least-squares approach to the evaluation of protein structures computed from NMR data

Summary A method is proposed for defining a probability distribution on an ensemble of protein conformations from a 2D NOE spectrum, while at the same time back-calculating the experimental spectrum from the ensemble. This enables one to assess the relative quality and significance of the conformations, and to test the consistency of the ensemble as a whole with the experimental spectrum. The method eliminates the need to integrate the cross-peak intensities and is surprisingly insensitive to random noise in the spectrum. In this communication, these advantages are demonstrated by applying the method to simulated data, for which the correct result is already known.Abbreviations NOE nuclear Overhauser effect - BPTI basic pancreatic trypsin inhibitor - rmsd root-mean-square deviation     

A computer-based protocol for semiautomated assignments and 3D structure determination of proteins

Summary A strategy is presented for the semiautomated assignment and 3D structure determination of proteins from heteronuclear multidimensional Nuclear Magnetic Resonance (NMR) data. This approach involves the computer-based assignment of the NMR signals, identification of distance restraints from nuclear Overhauser effects, and generation of 3D structures by using the NMR-derived restraints. The protocol is described in detail and illustrated on a resonance assignment and structure determination of the FK506 binding protein (FKBP, 107 amino acids) complexed to the immunosuppressant, ascomycin. The 3D structures produced from this automated protocol attained backbone and heavy atom rmsd of 1.17 and 1.69 Å, respectively. Although more highly resolved structures of the complex have been obtained by standard interpretation of NMR data (Meadows et al. (1993) Biochemistry, 32, 754–765), the structures generated with this automated protocol required minimal manual intervention during the spectral assignment and 3D structure calculations stages. Thus, the protocol may yield an approximate order of magnitude reduction in the time required for the generation of 3D structures of proteins from NMR data.     

The three-dimensional structure of acyl-coenzyme A binding protein from bovine liver: Structural refinement using heteronuclear multidimensional NMR spectroscopy

Summary The 3D structure of bovine recombinant acyl-coenzyme A binding protein has been determined using multidimensional heteronuclear magnetic resonance spectroscopy in a study that combines investigations of ¹⁵N-labeled and unlabeled protein. The present structure determination is a refinement of the structure previously determined (Andersen, K.V. and Poulsen, F.M. (1992) J. Mol. Biol., 226, 1131–1141). It is based on 1096 distance restraints and 124 dihedral angle restraints of which 69 are for -angles and 8 for chiral centers and 47 for prochiral centers. The new experimental input for the structure determination has provided an increase of 263 distance restraints, 5 -angle restraints, and 32 -angle restraints in 2 chiral centers, and 31 prochiral centers restraining an additional 23 ¹, 8 ², and 1 ³ angles. The increase of 300 distance and dihedral angle restraints representing an additional 30% of input parameters for the structure determination has been shown to be in agreement with the first structure. A set of 29 structures has been calculated and each of the structures has been compared to a mean structure to give an atomic root mean square deviation of 0.44±0.12 (1 is 0.1 nm) for the backbone atoms C, C, and N in the four -helices A1, residues 4–15, A2, residues 21–36, A3, residues 51–62 and A4, residues 65–84. The loop-region of residues Gly⁴⁵-Lys⁵⁰ could not be defined by the restraints obtained by NMR.The program PRONTO has been used for the spectrum analysis, assignment of the individual nuclear Overhauser effects, the integration of the cross peaks, and the measurement of the coupling constants. The programs DIANA, X-PLOR and INSIGHT have been used in the structure calculations and evaluations.     

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by ³¹P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Comparison of distance matrices in studies of population structure and genetic microdifferentiation: quadratic assignment

Questions concerning the relative effects of various evolutionary forces in molding the genetic variability exhibited by groups of human populations have typically been investigated by comparing a variety of genetic and cultural/historical "distance" matrices. A major methodological difficulty has been the lack of formal testing procedures with which to assess the degree of confirmation or disconfirmation of an estimated measure of relationship between such matrices. In this paper, we examine a very flexible matrix combinatorial procedure which generates statistical significance levels for correlational measures of pattern similarity between distance matrices. A recent generalization of the basic procedure to the three-matrix case allows questions concerning which of two matrices best fits a third matrix to be formally tested. Applications of these hypothesis testing and inference procedures to two separate sets of genetic, geographic, and cultural distance matrices illustrates their potential for finally solving a long-standing problem in anthropological genetics.     

Calculation of protein conformations by proton-proton distance constraints. A new efficient algorithm

We have developed a method to determine the three-dimensional structure of a protein molecule from such a set of distance constraints as can be determined by nuclear magnetic resonance studies. The currently popular methods for distance geometry based on the use of the metric matrix are applicable only to small systems. The method developed here is applicable to large molecules, such as proteins, with all atoms treated explicitly. This method works in the space of variable dihedral angles and determines a three-dimensional structure by minimization of a target function. We avoid difficulties hitherto inherent in this type of approach by two new devices: the use of variable target functions; and a method of rapid calculation of the gradient of the target functions. The method is applied to the determination of the structures of a small globular protein, bovine pancreatic trypsin inhibitor, from several artificial sets of distance constraints extracted from the X-ray crystal structure of this molecule. When a good set of constraints was available for both short- and long-range distances, the crystal structure was regenerated nearly exactly. When some ambiguities, such as those expected in experimental information, are allowed, the protein conformation can be determined up to a few local deformations. These ambiguities are mainly associated with the low resolving power of the short-range information.     

一株新型高产PHB假单胞菌SCH17的分离和特征分析

通过丁醇富集筛选,从土壤样品中筛选到一株菌株SCH17。经过生理特性和16S rRNA分析,鉴定菌株SCH17属于假单胞菌属。透射电镜显示该菌细胞内聚集了大量颗粒状物质,经过氯仿抽提和核磁共振分析,确认这些颗粒物质是聚β-羟基丁酸(PHB)。通过对碳源和氮源的优化,得到最佳积累PHB的碳源是果糖,氮源是蛋白胨。在该培养基中仅需发酵14 h,菌体干重和PHB含量均达到最大,分别为3.52 g/L和2.69 g/L,PHB含量高达细胞干重的76%。     

MICE: multiple-peak identification, characterization, and estimation

MICE--multiple-peak identification, characterization, and estimation--is a procedure for estimating a lower bound of the number of frequency peaks and for estimating the frequency peak parameters. The leading application is protein structure determination using nuclear magnetic resonance (NMR) experiments. NMR frequency data are multiple-peak data, where each frequency peak corresponds to two connected atoms in the three-dimensional protein structure. We analyze the NMR frequency data through a series of steps: a preliminary step for separating the signal from the background followed by identification of local maxima up to a noise-level-dependent threshold, estimation of the frequency peak parameters using an iterative algorithm, and detection of mixtures of peaks using hypothesis testing.     

A tetrameric allyl complex of sodium, and computational modeling of the Na-allyl chemical shift

Transmetallation of Li[A′] (A′ = [1,3-(SiMe₃)₂C₃H₃]⁻) with sodium tert-butoxide produces the corresponding sodium salt, which crystallizes from THF/toluene in the form of a cyclic tetramer, {Na[A′](thf)}₄. The Na atoms are in a square planar arrangement, bridged with π-bound allyl ligands; the Na-C distances range from 2.591(3)-2.896(3) Å, with an average of 2.70 Å. The geometries of several model organosodium complexes containing cyclopentadienyl and allyl ligands were optimized with density functional theory methods. The optimized structures were used with the gauge-including atomic orbital (GIAO) method to calculate their ²³Na NMR magnetic shielding values. Unlike the case with NaCp, the chemical shift of unsubstituted Na(C₃H₅) is very sensitive to the presence of coordinated THF (causing a 20 ppm upfield shift); silyl substitution has an even larger effect (30 ppm upfield shift). The observed ²³Na shift of δ −3.3 ppm for Na[A′] in THF-d₈, however, cannot be reliably distinguished from that calculated for the [Na(thf)₄]⁺ cation alone.     

Alloprenols: novel alpha-trans-polyprenols of Allophylus caudatus

A novel type of polyprenols, alloprenols, with an α-trans-isoprenoid unit was found in the leaves of Allophylus caudatus (Sapindaceae) besides typical α-cis-polyprenols. The polyprenol family (Prenol-11-13, Prenol-12 dominating) was accompanied by traces of dolichols of the same chain-length. Prenol α-cis- and α-trans-isomers were chromatographically separated and their structure was analyzed by HPLC/ESI-MS, HR-ESI-MS and ¹H and ¹³C NMR spectroscopy. Model compounds, semi-synthetic α-isomers of all-trans-Pren-9 and mainly-cis-Pren-11, were obtained using an oxidation-reduction procedure. Comparison of their NMR spectra confirmed the structure of the newly identified polyprenols. The observed pattern of NMR signal shifts may be applied for elucidation of isoprenoid structure.     

Search for novel neuraminidase inhibitors: Design, synthesis and interaction of oseltamivir derivatives with model membrane using docking, NMR and DSC methods

As a part of our ongoing program of developing novel influenza virus inhibitors, some new derivatives of oseltamivir were prepared by modifying the amino group with glycyl, acetyl, benzyl and prolyl moieties. The interactions of these derivatives with neuraminidase have been probed by molecular modeling techniques. Further, the interaction of these derivatives with model membranes prepared from DPPC and the effect on the thermotropic behavior and polymorphism of the bilayers have been investigated by multinuclear NMR and DSC methods. Results indicate that the glycyl derivative of oseltamivir has the most profound effects on the membrane, compared to other derivatives and seems to be the most promising derivative for further pharmacological evaluation as a neuraminidase inhibitor.     

Polyaminooligonucleotide: NMR structure of duplex DNA containing a nucleoside with spermine residue,N-[4,9,13-triazatridecan-1-yl]-2′-deoxycytidine

Background

The nature of the polyamine–DNA interactions at a molecular level is not clearly understood.

Methods

In order to shed light on the binding preferences of polyamine with nucleic acids, the NMR solution structure of the DNA duplex containing covalently bound spermine was determined.

Results

The structure of 4-N-[4,9,13-triazatridecan-1-yl]-2′-deoxycytidine (dCSp) modified duplex was compared to the structure of the reference duplex. Both duplexes are regular right-handed helices with all attributes of the B-DNA form. The spermine chain which is located in a major groove and points toward the 3′ end of the modified strand does not perturb the DNA structure.

Conclusion

In our study the charged polyamine alkyl chain was found to interact with the DNA surface. In the majority of converged structures we identified the presumed hydrogen bonding interactions between O6 and N7 atoms of G4 and the first internal –NH2⁺− amino group. Additional interaction was found between the second internal –NH2⁺− amino group and the oxygen atom of the phosphate of C3 residue.

General significance

The knowledge of the location and nature of a structure-specific binding site for spermine in DNA should be valuable in understanding gene expression and in the design of new therapeutic drugs.     

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.     

Binding mechanism of trans-N-caffeoyltyramine and human serum albumin: Investigation by multi-spectroscopy and docking simulation

trans-N-Caffeoyltyramine (TNC), which was isolated from the Cortex Lycii in our laboratory, is a phenolic amide compound with multiple pharmacological activities. The interaction between TNC and human serum albumin (HSA) was studied by Nuclear magnetic resonance (NMR) relaxation experiment, fluorescence spectroscopy, and docking simulation. NMR methodology is based on the analysis of selective and non-selective spin-lattice relaxation rate enhancements of TNC protons in the presence of the HSA. Result indicated that the interaction occurred between HSA and TNC, and changed the proton magnetic environment of TNC. Fluorescence spectroscopy confirmed that TNC displayed a strong capability to quench the fluorescence of HSA, and the acting forces for binding were hydrogen bonds and van der Waals forces. Furthermore, the circular dichroism, synchronous, and three-dimensional fluorescence spectra, which were employed to determine the conformation of protein, revealed that binding of TNC with HSA could induce conformational changes in HSA. In addition, the molecular modeling results exhibited that TNC mainly bonded to site I in sub-domain IIA of HSA.