Inter-residue Distances Research Articles

Kinetic description of structural changes linked to membrane import of the colicin E1 channel protein.

Upon binding to membranes, the 178-residue colicin E1 C-terminal channel protein forms a steady-state closed-channel intermediate that is a flexible extended two-dimensional helical array [Zakharov et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4282-4287]. Analysis of the kinetics of binding-insertion to liposome membranes of the channel protein, P178, and of changes of spectral parameters associated with structure transitions allowed a correlation of the sequence of tertiary and secondary structure changes with binding-insertion. Binding and insertion were distinguished by use of lipids modified with quenchers of Trp fluorescence attached to lipid headgroups or acyl chains. Secondary and tertiary structure changes were inferred, respectively, from changes in far-UV circular dichroism and relative changes of interresidue distances by fluorescence resonance energy transfer (FRET). "Single Trp" mutants were used in FRET analysis, with the background Tyr contribution determined through use of a "zero Trp" mutant. The sequence of distinguishable events and the pseudo-first-order rate constants under "standard" conditions (large unilamellar vesicles, pH 4.0, I = 0.1 M) was binding (30 +/- 5 s(-)(1)) --> unfolding (12.6 +/- 0.5 s(-)(1)) --> helix elongation (9.0 +/- 1.0 s(-)(1)) --> insertion (6. 6 +/- 0.5 s(-)(1)). Thus, helix elongation on the surface of the membrane can occur after unfolding and does not require insertion. Binding-insertion and structural transitions of P178 occur significantly faster with small unilamellar vesicles. The relevance to general mechanisms of protein import of the structural changes associated with import of the colicin channel is discussed.

Protein Fold Determination from Sparse Distance Restraints: The Restrained Generic Protein Direct Monte Carlo Method

The authors present the generate-and-select hierarchy for tertiary protein structure prediction. The foundation of this hierarchy is the Restrained Generic Protein (RGP) Direct Monte Carlo method. The RGP method is a highly efficient off-lattice residue buildup procedure that can quickly generate the complete set of topologies that satisfy a very small number of interresidue distance restraints. For three restraints uniformly distributed in a 72-residue protein, the authors demonstrate that the size of this set is {approximately}10{sup 4}. The RGP method can generate this set of structures in less than 1 h using a Silicon Graphics R10000 single processor workstation. Following structure generation, a simple criterion that measures the burial of hydrophobic and hydrophilic residues can reliably select a reduced set of {approximately}10{sup 2} structures that contains the native topology. A minimization of the structures in the reduced set typically ranks the native topology in the five lowest energy folds. Thus, using this hierarchical approach, the authors suggest that de novo prediction of moderate resolution globular protein structure can be achieved in just a few hours on a single processor workstation.

Crystal state and solution conformation of the B blood group trisaccharide alpha- l-Fucp-(12)-[alpha- d-Galp]-(13)]-beta- d-Galp-OCH3

The crystal structure of the human B blood group related trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)-beta-D-Galp-OCH3 (1) has been determined. The solution structure of 1 was studied by two-dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles phiH and psiH, derived from 3JCH coupling constants, and 10 inter-residue proton-proton distances. 3JCH could be accurately measured by a recently introduced two-dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement-derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo-Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related alpha-L-Fucp-(1-->2)-[6-deoxy-alpha-D-Galp]-(1-->3)-beta-D-Galp O-(CH2 )7CH3 trisaccharide (2).

Recent advances in site-directed spin labeling of proteins

Site-directed spin labeling of proteins is experiencing a phase of rapid technical evolution, application and evaluation. New strategies have been introduced for determining membrane protein topography, electrostatic potentials, the orientation of proteins at membrane surfaces and inter-residue distances. New applications include studies of β strands, structure mapping using spin—spin interactions, domain motions in soluble proteins and extensive structural analysis of a number of membrane and soluble proteins.

Distance geometry generates native-like folds for small helical proteins using the consensus distances of predicted protein structures.

For successful ab initio protein structure prediction, a method is needed to identify native-like structures from a set containing both native and non-native protein-like conformations. In this regard, the use of distance geometry has shown promise when accurate inter-residue distances are available. We describe a method by which distance geometry restraints are culled from sets of 500 protein-like conformations for four small helical proteins generated by the method of Simons et al. (1997). A consensus-based approach was applied in which every inter-Calpha distance was measured, and the most frequently occurring distances were used as input restraints for distance geometry. For each protein, a structure with lower coordinate root-mean-square (RMS) error than the mean of the original set was constructed; in three cases the topology of the fold resembled that of the native protein. When the fold sets were filtered for the best scoring conformations with respect to an all-atom knowledge-based scoring function, the remaining subset of 50 structures yielded restraints of higher accuracy. A second round of distance geometry using these restraints resulted in an average coordinate RMS error of 4.38 A.

Dynamic contact maps of protein structures

The two-dimensional contact map of interresidue distances is a visual analysis technique for protein structures. We present two standalone software tools designed to be used in combination to increase the versatility of this simple yet powerful technique. First, the program Structer calculates contact maps from three-dimensional molecular structural data. The contact map matrix can then be viewed in the graphical matrix-visualization program Dotter. Instead of using a predefined distance cutoff, we exploit Dotter’s dynamic rendering control, allowing interactive exploration at varying distance cutoffs after calculating the matrix once. Structer can use a number of distance measures, can incorporate multiple chains in one contact map, and allows masking of user-defined residue sets. It works either directly with PDB files, or can use the MMDB network API for reading structures.

Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus.

Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S. elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.

Facile synthesis of a high-affinity ligand for mammalian hepatic lectin containing three terminal N-acetylgalactosamine residues.

A simple cluster glycoside containing three residues of N-acetylgalactosamine with proper inter-residual distances can be a high-affinity ligand for asialoglycoprotein receptor of mammalian liver. YEE(ah-GalNAc)3 [Lee, R. T., and Lee, Y. C. (1987) Glycoconjugate J, 4, 317-328] is such a ligand having a Kd in the subnanomolar range, and this high-affinity ligand has been successfully utilized in the delivery of gene to the parenchymal cells of the liver [Merwin, J. R., Noell, G. S., Thomas, W. L., Chiou, H. C., DeRome, M. E., McKee, T. D., Spitalny, G. L., and Findeis, M. A. (1994) Bioconjugate Chem. 5, 612-620; Hangeland, J. J., Levis, J. T., Lee, Y. C., and Ts'o, P. O. P. (1995) Bioconjugate Chem. 6, 695-701]. Reported here is a synthetic procedure for an equally effective, homologous trivalent ligand, YDD(G-ah-GalNAc)3. The advantage offered by this new cluster glycoside is that the synthetic scheme accomplishes purification of reaction intermediates and the product without chromatographic separations. This greatly simplifies the procedure and allows scale-up of the operation at reduced cost of production.

Conformation-invariant structures of the α1β1 human hemoglobin dimer

Analysis of the conformational differences between the oxy and deoxy forms of hemoglobin is complicated by shifting coordinate systems and correlated motions between different parts of the molecule. Methods independent of any frame of reference were used to study the differences in structure between the oxy and deoxy forms of the human hemoglobin αβ dimer. Differences between the deoxy and oxy dimer structures can be characterized as rearrangements of 15 substructures persisting between the two conformations. Such substructures are of two kinds, either rigid domains or tertiary substructures. Rigid domains are groups of residues for which all inter-residue distances are conformationally invariant. Residues belonging to a rigid domain do not have to be spatially contiguous nor must they have consecutive sequence numbers. The largest such substructure is a rigid core that spans both the α and β monomers and includes 44% of the dimer. Other rigid domains exist within the heme pockets.An alternative but closely related view of the molecule is based on tertiary substructures. Unlike a rigid domain, a tertiary substructure must have consecutively numbered residues and the residue that ends one tertiary substructure begins the next. The decomposition of the dimer into tertiary substructures represents the dimer as a framework of connected stiff structural elements. Viewed as a set of tertiary substructures, the hemoglobin dimer has the same three principal functional elements: the dimer core and the α and β heme pockets, with the heme pockets held to the dimer core by CD and FG corners. The tertiary substructures that comprise the dimer core include 51% of the molecule. When ligands bind at the hemes, the FG corners communicate structural changes in the hemes to the dimer cores, which may mediate heme-heme cooperativity.

Three-Dimensional Structural Feature Search of Proteins

Abstract This paper describes a three-dimensional (3D) structural feature search, or motif search, of proteins using SS3D-P. Our present approach is based upon the use of a substructure searching program for small organic molecules, SS3D, reported in our previous work. In SS3D-P, the 3D structure of a protein is approximated with the coordinates of α-carbon atoms of the main chain, and treated as a molecular graph that is represented using a distance matrix. The program allows us to specify the tolerance value of the inter-residual distances to be compared. Several characterization schemes are also available for the different amino acid residues that can be distinguished. The program we have developed was validated by search trials with 3D query patterns of known protein motif structures: the P-loop, the EF-hand, and the Zinc finger motif using Protein Data Bank (PDB) structure files.

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.

Solution structure of neuropeptide tyrosine 13-36, a Y2 receptor agonist, as determined by NMR.

The three-dimensional structure of neuropeptide tyrosine (NPY) 13-36, a specific Y2 receptor agonist, has been investigated by two-dimensional 1H-NMR spectroscopy in solution. Analysis of the double-quantum-filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) spectra provided a complete assignment of the proton signals. The interproton connectivities observed in the NOESY spectra comprised 166 intraresidue and 95 interresidue distance ranges which were used as constraints for molecular modeling by distance geometry, simulated annealing and energy minimization. The optimal structures are characterized by a helical C-terminal fragment Leu30-Tyr36 and a wide loop from Leu17 to Ser22. The structure of NPY 13-36 is analogous to the structure of NPY under the same solvent conditions. Comparison with other reported Y2 agonists suggests that the helical Leu30-Tyr36 fragment is the most critical for activity.

Sampling protein conformations using segment libraries and a genetic algorithm

We present a new simulation algorithm for minimizing empirical contact potentials for a simplified model of protein structure. The model consists of backbone atoms only (including Cβ) with the φ and ψ dihedral angles as the only degrees of freedom. In addition, φ and ψ are restricted to a finite set of 532 discrete pairs of values, and the secondary structural elements are held fixed in ideal geometries. The potential function consists of a look-up table based on discretized inter-residue atomic distances. The minimization consists of two principal elements: the use of preselected lists of trial moves and the use of a genetic algorithm. The trial moves consist of substitutions of one or two complete loop regions, and the lists are in turn built up using preselected lists of randomly-generated three-residue segments. The genetic algorithm consists of mutation steps (namely, the loop replacements), as well as a hybridization step in which new structures are created by combining parts of two “parents’’ and a selection step in which hybrid structures are introduced into the population. These methods are combined into a Monte Carlo simulated annealing algorithm which has the overall structure of a random walk on a restricted set of preselected conformations. The algorithm is tested using two types of simple model potential. The first uses global information derived from the radius of gyration and the rms deviation to drive the folding, whereas the second is based exclusively on distance-geometry constraints. The hierarchical algorithm significantly outperforms conventional Monte Carlo simulation for a set of test proteins in both cases, with the greatest advantage being for the largest molecule having 193 residues. When tested on a realistic potential function, the method consistently generates structures ranked lower than the crystal structure. The results also show that the improved efficiency of the hierarchical algorithm exceeds that which would be anticipated from tests on either of the two main elements used independently.

Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling.

T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H.R., & Matthews, B.W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 A relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X.J., Wozniak, J.A., & Matthews, B.W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.

A New Approach to Clustering the Amino Acid

Each amino acid is represented by a vector of numerical measurements for the attributes of volume, area, hydrophilicity, polarity, hydrogen bonding, shape, and charge. Inter-residue distances are then calculated according to common metrics, and we introduce a new clustering objective function derived from information-theoretic considerations. The arguments of the function are the inter-object distances of the things to be clustered: in this case the amino acids. By means of approximating the solution of an integer programming problem, then, the residues are partitioned into clusters. The clusters obtained are compared with groups obtained in substitution/mutation studies and found to be similar. Thus, probably the strongest and most objective evidence to date is supplied for believing that physico-chemical properties account for the viability of substitutions and that the important similarities/differences are explained by a relatively small and simple set of properties.

Characterization of global molecular shape transitions between “open” and “closed” protein conformations

Induced-fit configurational transitions in proteins can take many forms. In cases, we find small “closures” of a loop onto the substrate. In other cases, the structural changes triggered by a ligand involve large rearrangements that affect entire domains. The nature of these transitions is normally assessed by a visual analysis or in terms of simple local geometrical parameters, such as interresidue distances, backbone dihedral angles, and relative displacements between domains. This approach is limited and rather undiscriminating. In this work, we apply recently introduced ideas from macromolecular shape analysis to characterize the global shape changes accompanying “open closed” transitions in proteins. Here, we monitor two distinct properties simultaneously: molecular size and self-entanglements. The method is applied to some proteins exhibiting pairs of structurally different conformations (adenylate kinase, hexokinase, citrate synthase, alcohol dehydrogenase, triosephosphate isomerase, thioredoxin, and aspartate amino-transferase). The conformational change associated with these proteins is classified according to an order parameter that considers various molecular shape features. The results allow one to recognize, in a nonvisual fashion, the likely occurrence of local or global structural rearrangements. In addition, the technique provides an insight into folding features that may remain invariant during the configurational transitions. © 1996 John Wiley & Sons, Inc.

Structure and Conversion Kinetics of a Bi-stable DNA i-motif: Broken Symmetry in the [d(5mCCTCC)]4 Tetramer

At slightly acidic pH, protonation of C-rich oligomers results in the formation of a four-stranded structure composed of two parallel duplexes in a head to tail orientation with their hemi-protonated C·C +pairs intercalated in a so-called i-motif. In all cases reported previously the duplexes are identical. The tetramer formed by the d(5mCCTCC) oligomer is different. The structure is computed on the basis of 55 inter-residue distances derived from NOESY cross-peaks measured at short mixing times. It consists of two intercalated non-equivalent symmetrical duplexes. The base stacking order is C5* C1 C4* C2 (T3*) T3 C2* C4 C1* C5, but the thymidine bases (T3*) of one duplex are looped out and lie in the wide grooves of the tetramer. The thymidine bases T3 stack as a symmetrical T·T pair between the sequentially adjacent C2·C2 +pair and the C2*·C2* +pair of the other duplex. Numerous exchange cross-peaks provide evidence for duplex intercon version. The interconversion rate is 1.4 s −1at 0°C and the activation energy is 94 kJ/mol. The opening of the T3·T3 pair, the closing of the T3*·T3 pair, and the opening of the C2*·C2* +pair occur simultaneously with the duplex interconversion. This suggests that the concerted opening and closing of the thymidine bases drive the duplex interconversion. Opening of the C4·C4 +and C4*·C4* +pairs, and dissociation of the tetramer are not part of the interconversion since they occur at much slower rates. Duplex interconversion within the [d(5mCCTCC)]4 tetramer provides the first structural and kinetics characterization of broken symmetry in a biopolymer. The tetramer formed by d(5mCCUCC) adopts a similar structure, but the rate of duplex interconversion is faster: 40 s −1at 0°C. At 32°C, interconversion is fast on the NMR time scale.

Watching proteins move using site-directed spin labeling

Site-directed spin labeling of proteins has proven to be a practical means for determining secondary structure and its orientation; surfaces of tertiary interactions; inter-residue distances; chain topology and depth of a given side chain from the membrane/aqueous surface in membrane proteins; and local electrostatic potentials at solvent-exposed sites. Moreover, the mobility of a side chain together with its solvent-accessibility may serve to uniquely identify the topographical location of specific residues in the protein fold. Future spectral analysis should permit a quantitative estimation of the contribution of backbone flexibility to the overall side-chain dynamics. The ability to time-resolve the structural features mentioned above makes SDSL a powerful approach for exploring the evolution of structure on the millisecond time scale. We anticipate future applications to the study of protein folding both in solution and in chaperone-mediated systems.

NMR-Based, Molecular Dynamics- and Random Walk Molecular Mechanics-Supported Study of Conformational Aspects of a Carbohydrate Ligand (Galβ1-2Galβ1-R) for an Animal Galectin in the Free and in the Bound State

The binding of a carbohydrate to a lectin may affect the conformation of the ligand. To address this question for the galectin from chicken liver, the conformation of Galβ1-R was analyzed in the free and in the galectin-bound state with 2D-ROESY- and 1D- as well as 2D-transferred NOE-experiments. A computer-assisted analysis of spatial parameters of the ligand by molecular dynamics (MD) and random walk molecular mechanics (RAMM) calculations, taking different dielectric constants from ϵ =1 to ϵ = 80 and various force fields into account, were instrumental to define the energetic minima of the free state. NMR-derived interresidual distance constraints enabled a conformational mapping. The two overlapping interresidual distance constraints obtained from transferred-NOE experiments of the galectin-ligand complex clearly support the notion that the conformation of the disaccharide in the bound state is at least very close to its global energy minimum state in solution.

Inter-C? atomic potentials derived from the statistics of average interresidue distances in proteins: Application to bovine pancreatic trypsin inhibitor

New effective potentials acting between pairs of residues in proteins are proposed based on statistics of average distances and standard deviations between Cα atoms of residues in protein tertiary structures. Gaussian functions are adopted as analytical forms of the potentials. A protein structure is modeled as a chain molecule with a fixed bond length connecting particles approximating the effects of amino acid residues. The potentials derived in this study are used for conformational sampling of trypsin inhibitor from bovine pancreas. Sampling is done with the Monte Carlo simulated annealing method. Sampled conformations can be classified into a few groups or structural classes, and one of these classes contains structures relatively close (with 7.8–8.7 Å root mean square [rms] deviation) to the X-ray structure. The native structure exhibits relatively low energy. These results denote a rather smooth landscape of the present potential energy surfaces. One class of classified structures contains nativelike structures, which suggests that the native structure can be predicted by further refinement of structures in this class. We discuss other properties and the effectiveness of the present potentials for description of protein structures. © 1996 by John Wiley & Sons, Inc.