Protein Backbone Conformation Research Articles

Solid-State NMR Determination of Peptide Torsion Angles: Applications of 2H-Dephased REDOR

The backbone conformation of peptides and proteins is completely defined by the torsion angles (φ,ψ,ω) of each amino acid residue along the polypeptide chain. We demonstrate a solid-state NMR method based on heteronuclear distance measurements for determining (φ,ψ) angles. Simple and reliable deuterium phase modulated pulses (PM5) reintroduce dipolar couplings between 2H and a spin-1/2 nucleus. Measuring the 13Ci-1{2Hiα} REDOR distance across a peptide bond results in the torsion angle φi as a consequence of the restricted geometry of the peptide backbone. The 15Ni+1{2Hiα} REDOR distance across a peptide bond defines the torsion angle ψi. This approach is demonstrated for both the 3-spin X{2H2}REDOR case of glycine and the 2-spin X{2H}REDOR case, represented by l-alanine, using two different tripeptides. It is shown that the technique can handle multiple sample conformations. PM5-REDOR decay curves of the ψ angle show distinctly different behaviors between α-helix and β-sheet backbone conformations.

Mutation of the surface valine residues 8 and 44 in the rubredoxin from Clostridium pasteurianum: solvent access versus structural changes as determinants of reversible potential.

The Pr(i) sidechains of two adjacent valine residues, V8 and V44, define the surface of the rubredoxin from Clostridium pasteurianum and control access to its Fe(S-Cys)4 active site. To assess the effect of systematic change of the steric bulk of the alkyl sidechains, eight single and three double mutant proteins have been isolated which vary G (H), A (Me), V (Pr(i)), L (Bu(i)) and I (Bu(s)) at those positions. X-ray crystal structures of the Fe(III) forms of the V44A and V44I proteins are reported. Positive shifts in reversible potential of up to 116 mV are observed and attributed to increased polarity around the Fe(S-Cys)4 site induced by (1) changes in protein backbone conformation driven by variation of the steric demands of the sidechain substituents and (2) changes in solvent access to the side-chains of ligands C9 and C42. Data for the V44A mutant show that a minor change in the steric requirements of a surface residue can introduce a NH...Sgamma hydrogen bond at the active site and lead to a shift in potential of + 50 mV.

Insight into the Secondary Structure of Non-native Proteins Bound to a Molecular Chaperone α-Crystallin: AN ISOTOPE-EDITED INFRARED SPECTROSCOPIC STUDY

alpha-Crystallin, the major lens protein, acts as a molecular chaperone by preventing the aggregation of proteins damaged by heat and other stress conditions. To characterize the backbone conformation of protein folding intermediates that are recognized by the chaperone, we prepared the uniformly (13)C-labeled alphaA-crystallin. The labeling greatly reduced the overlapping between the conformation-sensitive amide I bands of alpha-crystallin and unlabeled substrate proteins. This procedure has allowed us to gain insight into the secondary structure of alpha-crystallin-bound species, an understanding which has previously been unattainable. Analysis of the infrared spectra of two substrate proteins (gamma- and beta(L)-crystallins) indicates that heat-destabilized conformers captured by alpha-crystallin are characterized by a high proportion of native-like secondary structure. In contrast to the chaperone-bound species, the same proteins subjected to heat treatment in the absence of alpha-crystallin preserve very little native secondary structure. These data show that alpha-crystallin specifically recognizes very early intermediates on the denaturation pathway of proteins. These aggregation-prone species are characterized by native-like secondary structure but compromised tertiary interactions. The experimental approach described in this study can be further applied to probe the backbone conformation of proteins bound to chaperones other than alpha-crystallin.

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive 13C Labeling and Two-Dimensional Solid-State NMR

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive 13C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective 13C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-13C]glucose preferentially labels the ends of the side chains, while [2-13C]glycerol labels the Cα of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic–anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively 13C labeled protein were performed using 15N–13C 2D correlation spectroscopy. From the time dependence of the 15N–13C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective 13C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.

Vibrational spectroscopic analysis of a chymotrypsin inhibitor isolated from Schizolobium parahyba (Vell) Toledo seeds

Laser Raman and Fourier transform infrared spectroscopies were applied in the investigation of conformational features of a chymotrypsin inhibitor (SPC), inactive on trypsin, isolated from Schizolobium parahyba, a Leguminosae of the Cesalpinoidae family, found in tropical and subtropical regions. As a serine protease inhibitor, its importance is related to the control of proteolytic activity, which in turn is involved in a wide range of critically important biotechnological issues, such as blood coagulation, tumour cell growth, and plant natural defences against predators. SPC is a 20 kDa molecular mass monomeric protein, with two disulfide bonds. Its complete aminoacid primary sequence has not yet been determined. We analysed protein backbone conformation for the lyophylized solid and for an evaporated film, through Raman scattering and FTIR, respectively. The presence of significant amounts of disordered structures and of non-negligible contributions from α-helical and β-sheet structures were reckoned in both cases. The geometries of the disulfide bonds were defined: a gauche-gauche-gauche geometry was verified for one of the two bridges and a transient gauche-gauche-trans/trans-gauche-trans geometry has been indicated for the second one.Two out of the three tyrosine residues were shown to be in external location in the solid protein, as well as the only tryptophan residue.

The Ribbon of Hydrogen Bonds in the Three-Dimensional Structure of Globular Proteins

We report quantum mechanical computations and experimental evidence which suggest that the backbone conformation of globular proteins depends generally on the conservation of that part of the hydrogen bond network or ribbon which is joined, in general, directly to the backbone and is largely independent of the remainder of this whole network of hydrogen bonds. The familiar hydrogen bonds of the α helix and the β sheet form about one-half of this ribbon of hydrogen bonds. Both water molecules and hydrogen bonding side chain groups are involved in the formation of the ribbon.This view of the three-dimensional structure of globular proteins in terms of the `molecule' allows us to deal with the non-secondary structure as well as with the familiar secondary structure. It also suggests that the ribbon contains approximately the same number of hydrogen bonds within all three structures – the α helix, the β sheet and the coil – and that this is the reason for the ease of interconversion of these three structures.

An Empirical Correlation between Amide Deuterium Isotope Effects on 13Cα Chemical Shifts and Protein Backbone Conformation

Three-dimensional, triple resonance NMR techniques are described for measurement of two-bond (intraresidual) and three-bond (sequential) amide deuterium isotope effects on 13Cα chemical shifts. Measurements were carried out for uniformly 15N and 13C labeled human ubiquitin equilibrated in a 50% H2O/50% D2O mixed solvent. The three-bond isotope shift, 3ΔCα(ND), ranges from about 10−50 ppb, and, except for residues with positive φ angles and residues followed by Gly, its magnitude is described by 3ΔCα(ND) = 30.1 + 22.2 sin(ψ) ± 3.4 ppb. The two-bond isotope shift, 2ΔCα(ND), ranges from 70 to 116 ppb and is also dominated by the local backbone geometry at the Cα position: 2ΔCα(ND) = 93.1 + 10.1 sin(φ+62°) + 12.0 sin(ψ+42°) ± 4.1 ppb.

Investigation of molecular structure in solids by two-dimensional NMR exchange spectroscopy with magic angle spinning

An approach to the investigation of molecular structures in disordered solids, using two-dimensional (2D) nuclear magnetic resonance (NMR) exchange spectroscopy with magic angle spinning (MAS), is described. This approach permits the determination of the relative orientation of two isotopically labeled chemical groups within a molecule in an unoriented sample, thus placing strong constraints on the molecular conformation. Structural information is contained in the amplitudes of crosspeaks in rotor-synchronized 2D MAS exchange spectra that connect spinning sideband lines of the two labeled sites. The theory for calculating the amplitudes of spinning sideband crosspeaks in 2D MAS exchange spectra, in the limit of complete magnetization exchange between the labeled sites, is presented in detail. A new technique that enhances the sensitivity of 2D MAS exchange spectra to molecular structure, called orientationally weighted 2D MAS exchange spectroscopy, is introduced. Symmetry principles that underlie the construction of pulse sequences for orientationally weighted 2D MAS exchange spectroscopy are explained. Experimental demonstrations of the utility of 2D MAS exchange spectroscopy in structural investigations of peptide and protein backbone conformations are carried out on a model 13C-labeled tripeptide, L-alanylglycylglycine. The dihedral angles φ and ψ that characterize the peptide backbone conformation at Gly-2 are obtained accurately from the orientationally weighted and unweighted 2D 13C NMR exchange spectra.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface.

Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity. Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP. We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.

Impact of point mutations on the structure and thermal stability of ribonuclease T1 in aqueous solution probed by Fourier transform infrared spectroscopy.

We undertook a detailed comparative analysis of the infrared spectra of wild-type ribonuclease T1 and three mutants: two single mutants, Tyr-45-->Trp (Y45W) and Trp-59-->Tyr (W59Y), and a double mutant, Tyr-45-->Trp/Trp-59-->Tyr (Y45W/W59Y). These mutants were selected because they are known to affect the activity of the enzyme. The structural differences were evaluated by using peptide backbone and side-chain "marker" bands as conformation-sensitive monitors. All mutations lead to a decrease of the thermal transition temperature, though the mutation Tyr-45-->Trp affects the Tm to a lesser degree than the replacement of Trp-59 by Tyr, both in the single (W59Y) and in the double (Y45W/W59Y) mutant. Small changes in the protein backbone conformation and in the microenvironment of certain amino acids, induced by the point mutations, could be detected. In particular, we found subtle differences in the hydrogen bonding pattern of the beta-strands in the mutants W59Y and Y45W/W59Y, compared to that in wild-type RNase T1 and in the mutant Y45W. Practically identical spectra in the amide I region were obtained for the double mutant Y45W/W59Y and the single mutant W59Y, demonstrating that it is the change from Trp to Tyr in position 59 (located at the interface between the alpha-helix and a beta-strand) which affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the polypeptide backbone conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pseudodihedrals: Simplified protein backbone representation with knowledge‐based energy

Pairwise contact energies do not explicitly take protein secondary structure into account, and so provide an incomplete description of conformational energy. In order to construct a Hamiltonian that specifically relates to protein backbone conformations, a simplified backbone angle is used. The pseudodihedral angle (the torsion angle between planes defined by 4 consecutive alpha-carbon atoms) provides a simplified backbone representation and continues to manifest information about secondary-structure elements: the pseudo-Ramachandran plot contains helical and sheetlike regions. The distribution of pseudodihedral angles is highly sensitive to the identity of the central pair of amino acids. Therefore, a sequence-dependent, knowledge-based potential energy was found according to a quasichemical approximation. These functions form complementary additions to the contact potentials currently in use. This pseudodihedral potential greatly enhances the ability to design sequences that are specific to a given conformation and also improves the ability to discriminate a native conformation from many other conformations.

Finite‐state and reduced‐parameter representations of protein backbone conformations

AbstractThis article studies the representation of protein backbone conformations using a finite number of values for the backbone dihedral angles. We develop a combinatorial search algorithm that guarantees finding the global minima of functions over the configuration space of discrete protein conformations, and use this procedure to fit finite‐state models to the backbones of globular proteins. It is demonstrated that a finite‐state representation with a reasonably small number of states yields either a small root‐mean‐square error or a small dihedral angle deviation from the native structure, but not both at the same time. The problem can be resolved by introducing limited local optimization in each step of the combinatorial search. In addition, it is shown that acceptable approximation is achieved using a single dihedral angle as an independent variable in local optimization. Results for 11 proteins demonstrate the advantages and shortcomings of both the finite‐state and reduced‐parameter approximations of protein backbone conformations. © 1994 by John Wiley & Sons, Inc.

Conformational classification of short backbone fragments in globular proteins and its use for coding backbone conformations

An objective and systematic method of coding backbone conformations of proteins is developed. For this purpose polypeptide backbone is regarded to consist of two types of overlapping structural units, viz. a (φ, ψ) fragment (C i αC′ONC i+1 αC′ONC i+2 α), and a (ψ, φ) fragment (NC i αC′ONC i+1 αC′). By means of the principal component analysis, these (φ, ψ) and (ψ, φ) fragments are found to form five and six distinct clusters in respective high-dimensional conformational space with only a small number of exceptional unclassified fragments. Boundaries of clusters are defined by multi-dimensional ellipsoids. This classification of conformations of short backbone fragments is used to code protein backbone three-dimensional structures. In particular, conformations of peptide fragments consisting of four or six residues are coded and analyzed in detail. This structural code allows us to distinguish such features of protein backbone structures that are not retained in the usual secondary structural representation based on patterns of backbone hydrogen bonds, e.g., various types of four-residue turns. Similarity index, based on the number of different one-letter codes in a pair of structural codes, is a powerful measure of conformational similarity. When combined with another similarity measure, atomic root-mean-square distance, accurate similarity between a pair of conformations of fragments can be detected.

The use of 1JC?H? coupling constants as a probe for protein backbone conformation

Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (+/- 0.5 Hz) measurement of one bond JC alpha H alpha coupling constants in proteins that are uniformly enriched with 13C. An empirical phi,psi-surface is calculated which describes the deviation of 1JC alpha H alpha from its random coil value, using 203 1JC alpha H alpha values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which phi and psi are known with good precision from previous X-ray crystallographic studies. Residues in alpha-helical conformation exhibit positive deviations of 4-5 Hz, whereas deviations in beta-sheet are small and, on average, slightly negative. Data indicate that 1JC alpha H alpha depends primarily on psi, and that 1JC alpha H alpha may be useful as a qualitative probe for secondary structure. Comparison of 1JC alpha H alpha coupling constants measured in free calmodulin and in its complex with a 26-amino-acid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the 'central helix' is even more extensive than in free calmodulin.

An empirical correlation between 1JC.alpha.H.alpha. and protein backbone conformation

An empirical correlation between 1JC.alpha.H.alpha. and protein backbone conformation

Peptide group chemical shift computation

AbstractIt has been found that protein NH protons on top of a peptide group plane experience large upfield conformation shifts. The joint analysis of this effect and other known effects of the peptide group on proton chemical shifts has led to a two‐term empirical expression for peptide group proton chemical shift computation as a function of protein backbone conformation. Both terms are expressed by McConnell's point‐dipole shielding expressions, one referred to an axis perpendicular to the peptide plane and origin in the coordinate centre of the OCN atoms and the other referred to an axis along the carbonyl bond and origin close to the oxygen atom. Values for the two constants have been determined by least‐squares fitting of the C‐α, H and amide NH chemical shifts of the protein ubiquitin. As a cross‐check on the validity of the expression, the C‐α, H and NH shifts of ribonuclease and BPTI (basic pancreatic trypsin inhibitor) have been computed. The general agreement between the observed and computed shifts and the correlation coefficients found (0.72 on average) indicate that the expression accounts for the main physical effects of the protein peptide group on the proton chemical shifts. It is shown that, together with ring current shifts, the expression explains the main characteristics of the C‐α, H and amide NH chemical shifts in proteins.

Assembly of polypeptide and protein backbone conformations from low energy ensembles of short fragments: development of strategies and construction of models for myoglobin, lysozyme, and thymosin beta 4.

Recently we developed methods for the construction of knowledge-based mean fields from a data base of known protein structures. As shown previously, this approach can be used to calculate ensembles of probable conformations for short fragments of polypeptide chains. Here we develop procedures for the assembly of short fragments to complete three-dimensional models of polypeptide chains. The amino acid sequence of a given protein is decomposed into all possible overlapping fragments of a given length, and an ensemble of probable conformations is calculated for each fragment. The fragments are assembled to a complete model by choosing appropriate conformations from the individual ensembles and by averaging over equivalent angles. Finally a consistent model is obtained by rebuilding the conformation from the average angles. From the average angles the local variability of the structure can be calculated, which is a useful criterion for the reliability of the model. The procedure is applied to the calculation of the local backbone conformations of myoglobin and lysozyme whose structures have been solved by X-ray analysis and thymosin beta 4, a polypeptide of 43 amino acid residues whose structure was recently investigated by NMR spectroscopy. We demonstrate that substantial fractions of the calculated local backbone conformations are similar to the experimentally determined structures.

A lattice model for protein structure prediction at low resolution.

The prediction of the folded structure of a protein from its sequence has proven to be a very difficult computational problem. We have developed an exceptionally simple representation of a polypeptide chain, with which we can enumerate all possible backbone conformations of small proteins. A protein is represented by a self-avoiding path of connected vertices on a tetrahedral lattice, with several amino acid residues assigned to each lattice vertex. For five small structurally dissimilar proteins, we find that we can separate native-like structures from the vast majority of non-native folds by using only simple structural and energetic criteria. This method demonstrates significant generality and predictive power without requiring foreknowledge of any native structural details.

Estimation of accuracy in determining protein backbone conformations from NOE data and empirical φ, ψ probability distributions

Comprehensive computational experiments were carried out to examine systematically the potential for deriving protein backbone conformations using NOE data in conjunction with the probability distribution function of known backbone conformations of amino acid residues in proteins. It is shown that, in combination with the backbone conformational probability distribution, the measurement of NOE distances with moderate precision, consistent with the precision of modem NMR methods, is adequate to produce good local conformations of the protein backbone. The apparent need for much higher precision is not a fundamental property of the problem; it arose in previous treatments only from the mathematical statement of the problem and the approaches used. To demonstrate the improved precision resulting from combined use of NOE data and the φ, ψ probability distribution, a computer program, FISINOE, was developed, and control calculations with both simulated and experimental NOE data for bovine pancreatic trypsin inhibitor were performed. An ensemble of conformations obtained from the X-ray Protein Data Bank was used to obtain an approximate probability distribution for existing conformations of amino acid residues in proteins. A comprehensive statistical analysis of the results clearly shows that the set of sequential d connectivities (data on the presence or absence of sequential NOE cross peaks related to nearest-neighbor residues), combined with prior information about existing conformations, is sufficient to obtain a satisfactorily determined local conformation of the protein backbone in most cases; in some cases it would be helpful to use, as additional information, the cross-peak intensity between the NH and C αH intraresidual protons. Comparative studies indicate that the stability and accuracy in estimating the φ and ψ angles by the FISINOE program exceed those of other traditional approaches. The principal advantages of the FISINOE method are discussed, and the extension of this method for the determination of protein three-dimensional structures is outlined.

Prediction of protein backbone conformation based on seven structure assignments: Influence of local interactions

A method is developed to compute backbone tertiary folds from the amino acid sequence. In this method, the number of degrees of freedom is drastically reduced by neglecting side-chain flexibility, and by describing backbone conformations as combinations of only seven structural states. These are characterized by single values of the dihedral angles φ, ψ and ω, representing allowed conformations of the isolated dipeptide. We show that this restrictive model is none the less capable of describing native backbones to within acceptable deviations. Using our backbone description, potentials of mean force are derived from a database of known protein structures, based on statistical influences of single residues and residue pairs on the conformational states in their vicinity along the chain. This yields the force-field component due to local interactions, which is then used to predict lowest-energy conformations from any given amino acid sequence. The prediction algorithm does not require searching conformational space andis therefore extremely fast. Another important asset of our method is that it is able to compute not only the minimum energy conformation, but any number of lowest energy structures, whose relative preferences can be determined from the corresponding computed energy values. The performance of our procedure is tested on short peptides that are likely to be stabilized by local interactions. These include several helical structures and a hexapeptide with a β-bend conformation, corresponding to peptides shown to have relatively well-defined conformations in aqueous solution, and to protein segments believed to adopt their native conformation early during folding. In addition, several flexible peptides are analysed. Except for the problems encountered in predicting observed disulphide bridges in two of the flexible peptides, and in a somewhat larger fragment comprising residues 30 to 51 of bovine trypsin inhibitor, prediction results compare very favourably with experimental data. Potential applications of our procedure to protein modelling and its extension to protein folding are discussed.