Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy.

Distance measurements are performed between a pair of spin labels attached to nucleic acids using Pulsed Electron-Electron Double Resonance (PELDOR, also called DEER) spectroscopy which is a complementary tool to other structure determination methods in structural biology. The rigid spin label Ç, when incorporated pairwise into two helical parts of a nucleic acid molecule, allows the determination of both the mutual orientation and the distance between those labels, since Ç moves rigidly with the helix to which it is attached. We have developed a two-step protocol to investigate the conformational flexibility of flexible nucleic acid molecules by multi-frequency PELDOR. In the first step, a library with a broad collection of conformers, which are in agreement with topological constraints, NMR restraints and distances derived from PELDOR, was created. In the second step, a weighted structural ensemble of these conformers was chosen, such that it fits the multi-frequency PELDOR time traces of all doubly Ç-labelled samples simultaneously. This ensemble reflects the global structure and the conformational flexibility of the two-way DNA junction. We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge at the center. The kink and twist motions between both helical parts were quantitatively determined and showed high flexibility, in agreement with a Förster Resonance Energy Transfer (FRET) study on a similar bent DNA motif. The approach presented here should be useful to describe the relative orientation of helical motifs and the conformational flexibility of nucleic acid structures, both alone and in complexes with proteins and other molecules.

Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments.

Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.

Conformational stability and activity of p73 require a second helix in the tetramerization domain.

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.

NMR structure reveals intramolecular regulation mechanism for pheromone binding and release.

Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the transport of pheromones, which are a special class of odorants, is mediated by pheromone-binding proteins (PBPs), which occur at high concentrations in the sensillar lymph. The PBP from the silk moth Bombyx mori (BmPBP) undergoes a pH-dependent conformational transition between the forms BmPBP(A) present at pH 4.5 and BmPBP(B) present at pH 6.5. Here, we describe the NMR structure of BmPBP(A), which consists of a tightly packed arrangement of seven alpha-helices linked by well defined peptide segments and knitted together by three disulfide bridges. A scaffold of four alpha-helices that forms the ligand binding site in the crystal structure of a BmPBP-pheromone complex is preserved in BmPBP(A). The C-terminal dodecapeptide segment, which is in an extended conformation and located on the protein surface in the pheromone complex, forms a regular helix, alpha(7), which is located in the pheromone-binding site in the core of the unliganded BmPBP(A). Because investigations by others indicate that the pH value near the membrane surface is reduced with respect to the bulk sensillar lymph, the pH-dependent conformational transition of BmPBP suggests a novel physiological mechanism of intramolecular regulation of protein function, with the formation of alpha(7) triggering the release of the pheromone from BmPBP to the membrane-standing receptor.

NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque-competence, A beta(1-40)(ox) and A beta(1-42)(ox).

NMR studies of amyloid beta-peptides (A beta) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer's disease-related conformational polymorphism of A beta. In the aqueous medium, neither of the polypeptides A beta(1-40)(ox) or A beta(1-42)(ox) (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of A beta(1-40)(ox) and A beta(1-42)(ox) are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in A beta(1-42)(ox) when compared to A beta(1-40)(ox). The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that A beta(1-42)(ox) in aqueous solution has much higher plaque-competence than A beta(1-40)(ox).

NMR structure of the calreticulin P-domain.

The NMR structure of the rat calreticulin P-domain, comprising residues 189-288, CRT(189-288), shows a hairpin fold that involves the entire polypeptide chain, has the two chain ends in close spatial proximity, and does not fold back on itself. This globally extended structure is stabilized by three antiparallel beta-sheets, with the beta-strands comprising the residues 189-192 and 276-279, 206-209 and 262-265, and 223-226 and 248-251, respectively. The hairpin loop of residues 227-247 and the two connecting regions between the beta-sheets contain a hydrophobic cluster, where each of the three clusters includes two highly conserved tryptophyl residues, one from each strand of the hairpin. The three beta-sheets and the three hydrophobic clusters form a repeating pattern of interactions across the hairpin that reflects the periodicity of the amino acid sequence, which consists of three 17-residue repeats followed by three 14-residue repeats. Within the global hairpin fold there are two well-ordered subdomains comprising the residues 219-258, and 189-209 and 262-284, respectively. These are separated by a poorly ordered linker region, so that the relative orientation of the two subdomains cannot be precisely described. The structure type observed for CRT(189-288) provides an additional basis for functional studies of the abundant endoplasmic reticulum chaperone calreticulin.

Sequence-specific NMR assignment of proteins by global fragment mapping with the program MAPPER.

A new program, MAPPER, for semiautomatic sequence-specific NMR assignment in proteins is introduced. The program uses an input of short fragments of sequentially neighboring residues, which have been assembled based on sequential NMR connectivities and for which either the 13C(alpha) and 13C(beta) chemical shifts or data on the amino acid type from other sources are known. MAPPER then performs an exhaustive search for self-consistent simultaneous mappings of all these fragments onto the protein sequence. Compared to using only the individual mappings of the spectroscopically connected fragments, the global mapping adds a powerful new constraint, which results in resolving many otherwise intractable ambiguities. In an initial application, virtually complete sequence-specific assignments were obtained for a 110 kDa homooctameric protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus.

NMR structures of three single-residue variants of the human prion protein.

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121-230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrP(C)) that is formed by the C-terminal part of helix 3, with residues 218-230, and a loop of residues 166-172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized "protein X," and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121-230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized "conformational markers" could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166-172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166-172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

NMR structure of the sea urchin (Strongylocentrotus purpuratus) metallothionein MTA.

The three-dimensional structure of [(113)Cd7]-metallothionein-A (MTA) of the sea urchin Strongylocentrotus purpuratus was determined by homonuclear(1)H NMR experiments and heteronuclear [(1)H, (113)Cd]-correlation spectroscopy. MTA is composed of two globular domains, an N-terminal four-metal domain of the amino acid residues 1 to 36 and a Cd4Cys11cluster, and a C-terminal three-metal domain including the amino acid residues 37 to 65 and a Cd3Cys9cluster. The structure resembles the known mammalian and crustacean metallothioneins, but has a significantly different connectivity pattern of the Cys-metal co-ordination bonds and concomitantly contains novel local folds of some polypeptide backbone segments. These differences can be related to variations of the Cys sequence positions and thus emphasize the special role of the cysteine residues in defining the structure of metallothioneins, both on the level of the domain architecture and the topology of the metal-thiolate clusters.

NMR solution structure of the periplasmic chaperone FimC.

The NMR structure of the 205-residue periplasmic chaperone FimC is presented. This protein consists of two globular domains with immunoglobulin-like folds connected by a 15-residue linker peptide. The relative orientation of the two domains is defined by hydrophobic contacts and an interdomain salt bridge. FimC mediates the assembly of type-1 pili, which are filamentous surface organelles of uropathogenic Escherichia coli strains that enable the bacteria to attach to host cell surfaces and persist in macrophages. The availability of the NMR structure of FimC provides a new basis for rational design of drugs against infections by uropathogenic bacteria.

Automated peak picking and peak integration in macromolecular NMR spectra using AUTOPSY.

A new approach for automated peak picking of multidimensional protein NMR spectra with strong overlap is introduced, which makes use of the program AUTOPSY (automated peak picking for NMR spectroscopy). The main elements of this program are a novel function for local noise level calculation, the use of symmetry considerations, and the use of lineshapes extracted from well-separated peaks for resolving groups of strongly overlapping peaks. The algorithm generates peak lists with precise chemical shift and integral intensities, and a reliability measure for the recognition of each peak. The results of automated peak picking of NOESY spectra with AUTOPSY were tested in combination with the combined automated NOESY cross peak assignment and structure calculation routine NOAH implemented in the program DYANA. The quality of the resulting structures was found to be comparable with those from corresponding data obtained with manual peak picking.

Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND.

The new computer algorithm FOUND, which is implemented as an integrated module of the DYANA structure calculation program, is capable of performing systematic local conformation analyses by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based DYANA target function value. Sets of mutually dependent torsion angles, for example in ribose rings, are treated as a single degree of freedom. The results of the local conformation analysis include allowed torsion angle ranges and stereospecific assignments for diastereotopic substituents, which are then included in the input of a subsequent structure calculation. FOUND can be used for grid searches comprising up to 13 torsion angles, such as the backbone of a complete alpha-helical turn or dinucleotide fragments in nucleic acids, and yields a significantly higher number of stereospecific assignments than the precursor grid search algorithm HABAS.

PSEUDYANA for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics.

The program DYANA, for calculation of solution structures of biomolecules with an algorithm based on simulated annealing by torsion angle dynamics, has been supplemented with a new routine, PSEUDYANA, that enables efficient use of pseudocontact shifts as additional constraints in structure calculations of paramagnetic metalloproteins. PSEUDYANA can determine the location of the metal ion inside the protein frame and allows to define a single tensor of magnetic susceptibility from a family of conformers. As an illustration, a PSEUDYANA structure calculation is provided for a metal-undecapeptide complex, where simulated pseudocontact shifts but no NOE restraints are used as conformational constraints.

Torsion angle dynamics for NMR structure calculation with the new program DYANA.

The new program DYANA (DYnamics Algorithm for Nmr Applications) for efficient calculation of three-dimensional protein and nucleic acid structures from distance constraints and torsion angle constraints collected by nuclear magnetic resonance (NMR) experiments performs simulated annealing by molecular dynamics in torsion angle space and uses a fast recursive algorithm to integrate the equations of motions. Torsion angle dynamics can be more efficient than molecular dynamics in Cartesian coordinate space because of the reduced number of degrees of freedom and the concomitant absence of high-frequency bond and angle vibrations, which allows for the use of longer time-steps and/or higher temperatures in the structure calculation. It also represents a significant advance over the variable target function method in torsion angle space with the REDAC strategy used by the predecessor program DIANA. DYANA computation times per accepted conformer in the "bundle" used to represent the NMR structure compare favorably with those of other presently available structure calculation algorithms, and are of the order of 160 seconds for a protein of 165 amino acid residues when using a DEC Alpha 8400 5/300 computer. Test calculations starting from conformers with random torsion angle values further showed that DYANA is capable of efficient calculation of high-quality protein structures with up to 400 amino acid residues, and of nucleic acid structures.

The NMR solution conformation of unligated human cyclophilin A.

The nuclear magnetic resonance (NMR) solution structure of free, unligated cyclophilin A (CypA), which is an 18 kDa protein from human T-lymphocytes that was expressed in Escherichia coli for the present study, was determined using multidimensional heteronuclear NMR techniques. Sequence-specific resonance assignments for 99.5% of all backbone amide protons and non-labile hydrogen atoms provided the basis for collection of an input of 4101 nuclear Overhauser enhancement (NOE) upper distance constraints and 371 dihedral angle constraints for distance geometry calculations and energy minimization with the programs DIANA and OPAL. The average RMSD values of the 20 best energy-refined NMR conformers relative to the mean coordinates are 0.49 A for the backbone atoms and 0.88 A for all heavy atoms of residues 2 to 165. The molecular architecture includes an eight-stranded antiparallel beta-barrel that is closed by two amphipathic alpha-helices. Detailed comparisons with the crystal structure of free CypA revealed subtle but significant conformational differences that can in most cases be related to lattice contacts in the crystal structure. 15N spin relaxation times and NMR lineshape analyses for CypA in the free form and complexed with cyclosporin A (CsA) revealed transitions of polypeptide loops surrounding the ligand-binding site from locally flexible conformations in the free protein, some of which include well-defined conformational equilibria, to well-defined spatial arrangements in the CypA-CsA complex. Compared to the crystal structure of free CypA, where the ligand-binding area is extensively involved in lattice contacts, the NMR structure presents a highly relevant reference for studies of changes in structure and internal mobility of the binding pocket upon ligand binding, and possible consequences of this conformational variability for calcineurin recognition by the CypA-CsA complex.

Automated combined assignment of NOESY spectra and three-dimensional protein structure determination.

A procedure for automated protein structure determination is presented that is based on an iterative procedure during which the NOESY peak list assignment and the structure calculation are performed simultaneously. The input consists of a list of NOESY peak positions and a list of chemical shifts as obtained from sequence-specific resonance assignment. For the present applications of this approach the previously introduced NOAH routine was implemented in the distance geometry program DIANA. As an illustration, experimental 2D and 3D NOESY cross-peak lists of six proteins have been analyzed, for which complete sequence-specific 1H assignments are available for the polypeptide backbone and the amino acid side chains. The automated method assigned 70-90% of all NOESY cross peaks, which is on average 10% less than with the interactive approach, and only between 0.8% and 2.4% of the automatically assigned peaks had a different assignment than in the corresponding manually assigned peak lists. The structures obtained with NOAH/DIANA are in close agreement with those from manually assigned peak lists, and with both approaches the residual constraint violations correspond to high-quality NMR structure determinations. Systematic comparisons of the bundles of conformers that represent corresponding automatically and interactively determined structures document the absence of significant bias in either approach, indicating that an important step has been made towards automation of structure determination from NMR spectra.

The new program OPAL for molecular dynamics simulations and energy refinements of biological macromolecules.

A new program for molecular dynamics (MD) simulation and energy refinement of biological macromolecules, OPAL, is introduced. Combined with the supporting program TRAJEC for the analysis of MD trajectories, OPAL affords high efficiency and flexibility for work with different force fields, and offers a user-friendly interface and extensive trajectory analysis capabilities. Salient features are computational speeds of up to 1.5 GFlops on vector supercomputers such as the NEC SX-3, ellipsoidal boundaries to reduce the system size for studies in explicit solvents, and natural treatment of the hydrostatic pressure. Practical applications of OPAL are illustrated with MD simulations of pure water, energy minimization of the NMR structure of the mixed disulfide of a mutant E. coli glutaredoxin with glutathione in different solvent models, and MD simulations of a small protein, pheromone Er-2, using either instantaneous or time-averaged NMR restraints, or no restraints.