Ensemble Of NMR Structures Research Articles

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues.

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein-protein interactions. We studied aromatic residues in the two structurally homologous cross-β amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-β architecture.

A Novel Model of the Dimeric Human Hv1 Channel Reveals a Putative ATP Binding Site

A multi-template model of the human voltage-gated proton channel (hHv1) was developed by combining the recently solved NMR structural ensemble of the hHv1 (PDBID: 5OQK) with the coiled-coil C-terminal domain (PDBID: 3A2A), bridging them through the chimeric mHv1cc structure (PDBID: 3WKV). The model was built and refined as a dimer within Rosetta's Membrane framework, using the symmetry extracted from the coiled-coil and including experimentally derived constraints during the relaxation. The dimeric model was then subjected to extensive Gaussian Accelerated Molecular Dynamics simulations in a POPC bilayer, and representative snapshots were extracted from the trajectory by clustering analysis. Blind docking simulations of an ATP molecule on this structural ensemble revealed a putative ATP binding site located on the intracellular portion of the permeation pathway, below the hydrophobic gasket. The same region was identified by two sequence-based ATP binding site predictors: ATPSite and ATPint; and a third meta-predictor which combines sequence and structure-based features: ATPBind. According to the in-silico predictions, we found that whole-cell hHv1 current measured in presence of intracellular 3 mM ATP (patch-clamp, Jurkat T cells, n=7) showed a higher amplitude (148.3±27.0 vs 35.6±4.6 pA at 20 mV p<0.05) and a hyperpolarized Vthreshold (−25.7±2.0 vs −12.0±3.7 mV, p<0.05), compared to control cells (without ATP, n=5). The combination of several in-silico methodologies allowed us to develop a novel model of dimeric hHv1 channel, and predict its direct modulation by ATP. Moreover, experimental results confirmed that ATP is able to activate the channel. In a physiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation such as pancreatic β-cells, cancer cells or immune cells.

Analysis of the interface variability in NMR structure ensembles of protein-protein complexes.

NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html.

Sequence, structure, and cooperativity in folding of elementary protein structural motifs

Residue-level unfolding of two helix-turn-helix proteins--one naturally occurring and one de novo designed--is reconstructed from multiple sets of site-specific (13)C isotopically edited infrared (IR) and circular dichroism (CD) data using Ising-like statistical-mechanical models. Several model variants are parameterized to test the importance of sequence-specific interactions (approximated by Miyazawa-Jernigan statistical potentials), local structural flexibility (derived from the ensemble of NMR structures), interhelical hydrogen bonds, and native contacts separated by intervening disordered regions (through the Wako-Saitô-Muñoz-Eaton scheme, which disallows such configurations). The models are optimized by directly simulating experimental observables: CD ellipticity at 222 nm for model proteins and their fragments and (13)C-amide I' bands for multiple isotopologues of each protein. We find that data can be quantitatively reproduced by the model that allows two interacting segments flanking a disordered loop (double sequence approximation) and incorporates flexibility in the native contact maps, but neither sequence-specific interactions nor hydrogen bonds are required. The near-identical free energy profiles as a function of the global order parameter are consistent with expected similar folding kinetics for nearly identical structures. However, the predicted folding mechanism for the two motifs is different, reflecting the order of local stability. We introduce free energy profiles for "experimental" reaction coordinates--namely, the degree of local folding as sensed by site-specific (13)C-edited IR, which highlight folding heterogeneity and contrast its overall, average description with the detailed, local picture.

Structure and mechanism of the primosome protein DnaT-functional structures for homotrimerization, dissociation of ssDNA from the PriB·ssDNA complex, and formation of the DnaT·ssDNA complex.

In Escherichia coli, the primosome plays an essential role in replication restart after dissociation of replisomes at the damaged replication fork. As well as PriA and PriB, DnaT, an ssDNA-binding protein, is a key member of the primosome. In this study, limited proteolysis indicated that E. coli DnaT was composed of two domains, consistent with the results of recent studies using Klebsiella pneumonia DnaT. We also found that a specific 24-residue region (Phe42-Asp66) in the N-terminal domain (1-88) was crucial for DnaT trimerization. Moreover, we determined the structure of the DnaT C-terminal domain (89-179) by NMR spectroscopy. This domain included three α-helices and a long flexible C-terminal tail, similar to the C-terminal subdomain of the AAA+ ATPase family. The neighboring histidines, His136 and His137, at a position corresponding to the AAA+ sensor II motif, were suggested to form an ssDNA-binding site. Furthermore, we found that the acidic linker between the two domains had an activity for dissociating ssDNA from the PriB·ssDNA complexes in a manner supported by the conserved acidic residues Asp70 and Glu76. Thus, these findings provide a novel structural basis for understanding the mechanism of DnaT in exposure of ssDNA and reloading of the replicative helicase at the stalled replication fork. The coordinates used for the ensemble of NMR structures have been deposited in the Protein Data Bank under accession code 2ru8. The NMR data have been deposited in the BioMagResBank (www.bmrb.wisc.edu) under accession number 11549.

Structure of the Neisserial outer membrane protein Opa₆₀: loop flexibility essential to receptor recognition and bacterial engulfment.

The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen–host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.

FLAMEnGO 2.0: An enhanced fuzzy logic algorithm for structure-based assignment of methyl group resonances

We present an enhanced version of the FLAMEnGO (Fuzzy Logic Assignment of Methyl Group) software, a structure-based method to assign methyl group resonances in large proteins. FLAMEnGO utilizes a fuzzy logic algorithm coupled with Monte Carlo sampling to obtain a probability-based assignment of the methyl group resonances. As an input, FLAMEnGO requires either the protein X-ray structure or an NMR structural ensemble including data such as methyl–methyl NOESY, paramagnetic relaxation enhancement (PRE), methine–methyl TOCSY data. Version 2.0 of this software (FLAMEnGO 2.0) has a user-friendly graphic interface and presents improved modules that enable the input of partial assignments and additional NMR restraints. We tested the performance of FLAMEnGO 2.0 on maltose binding protein (MBP) as well as the C-subunit of the cAMP-dependent protein kinase A (PKA-C). FLAMEnGO 2.0 can be used as a standalone method or to assist in the completion of partial resonance assignments and can be downloaded at www.chem.umn.edu/groups/veglia/forms/flamengo2-form.html.

Designing Molecular Dynamics Simulations to Shift Populations of the Conformational States of Calmodulin

We elucidate the mechanisms that lead to population shifts in the conformational states of calcium-loaded calmodulin (Ca2+-CaM). We design extensive molecular dynamics simulations to classify the effects that are responsible for adopting occupied conformations available in the ensemble of NMR structures. Electrostatic interactions amongst the different regions of the protein and with its vicinal water are herein mediated by lowering the ionic strength or the pH. Amino acid E31, which is one of the few charged residues whose ionization state is highly sensitive to pH differences in the physiological range, proves to be distinctive in its control of population shifts. E31A mutation at low ionic strength results in a distinct change from an extended to a compact Ca2+-CaM conformation within tens of nanoseconds, that otherwise occur on the time scales of microseconds. The kinked linker found in this particular compact form is observed in many of the target-bound forms of Ca2+-CaM, increasing the binding affinity. This mutation is unique in controlling C-lobe dynamics by affecting the fluctuations between the EF-hand motif helices. We also monitor the effect of the ionic strength on the conformational multiplicity of Ca2+-CaM. By lowering the ionic strength, the tendency of nonspecific anions in water to accumulate near the protein surface increases, especially in the vicinity of the linker. The change in the distribution of ions in the vicinal layer of water allows N- and C- lobes to span a wide variety of relative orientations that are otherwise not observed at physiological ionic strength. E31 protonation restores the conformations associated with physiological environmental conditions even at low ionic strength.

Protein conformational dynamics of crambin in crystal, solution and in the trajectories of molecular dynamics simulations

Atomic displacement parameters--B factors of the eight crambin crystal structures obtained at 0.54-1.5 angstroms resolution and temperatures of 100-293K have been analyzed. The comparable contributions to the B factor values are the intramolecular motions which are modeled by the harmonic vibration calculations and derived from the molecular dynamics simulation (MD) as well as rigid body changes in the position of a protein molecule as a whole. In solution for the average NMR structure of crambin the amplitudes of the backbone atomic fluctuations of the most residues of the segments with the regular backbone conformations are close to the amplitudes of the small scale harmonic vibrations. For the same residues the probability of the medium scale fluctuations fixed by the hydrogen exchange method is very low. The restricted conformational mobility of those segments is coupled with the depressed amplitudes of the fluctuation changes of the tertiary structure registered by the residue accessibility changes in an ensemble of NMR structures that forms the average NMR structure of crambin. The amplitudes of temperature fluctuations of backbone atoms and the tertiary structure raise in the segment with the irregular conformations, turn and loops. In the same segments the amplitudes of the calculated harmonic vibrations also increase, but to a lesser extent and especially in the interhelical loop with the most strong and complicated fluctuation changes of the backbone conformation. In solution for the NMR structure in this loop the conformational transitions occur between the conformational substates separated by the energy barriers, but they are not observed even in the long 100 ns trajectories from the MD simulation of crambin. These strong local fluctuation changes of the structure may play a key role in the protein functioning and modern performance improvements in the MD simulation techniques are oriented to increase the probability of protein appearance in the trajectories from the MD simulations.

Simultaneous single-structure and bundle representation of protein NMR structures in torsion angle space

A method is introduced to represent an ensemble of conformers of a protein by a single structure in torsion angle space that lies closest to the averaged Cartesian coordinates while maintaining perfect covalent geometry and on average equal steric quality and an equally good fit to the experimental (e.g. NMR) data as the individual conformers of the ensemble. The single representative 'regmean structure' is obtained by simulated annealing in torsion angle space with the program CYANA using as input data the experimental restraints, restraints for the atom positions relative to the average Cartesian coordinates, and restraints for the torsion angles relative to the corresponding principal cluster average values of the ensemble. The method was applied to 11 proteins for which NMR structure ensembles are available, and compared to alternative, commonly used simple approaches for selecting a single representative structure, e.g. the structure from the ensemble that best fulfills the experimental and steric restraints, or the structure from the ensemble that has the lowest RMSD value to the average Cartesian coordinates. In all cases our method found a structure in torsion angle space that is significantly closer to the mean coordinates than the alternatives while maintaining the same quality as individual conformers. The method is thus suitable to generate representative single structure representations of protein structure ensembles in torsion angle space. Since in the case of NMR structure calculations with CYANA the single structure is calculated in the same way as the individual conformers except that weak positional and torsion angle restraints are added, we propose to represent new NMR structures by a 'regmean bundle' consisting of the single representative structure as the first conformer and all but one original individual conformers (the original conformer with the highest target function value is discarded in order to keep the number of conformers in the bundle constant). In this way, analyses that require a single structure can be carried out in the most meaningful way using the first model, while at the same time the additional information contained in the ensemble remains available.

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy

The broad spectrum of antibacterial activities of host defense cationic antimicrobial peptides (AMPs) arises from their ability to perturb membrane integrity of the microbes. The mechanisms are often thought to require assembly of AMPs on the membrane surface to form pores. However, three dimensional structures in the oligomeric form of AMPs in the context of lipid membranes are largely limited. Here, we demonstrate that a 22-residue antimicrobial peptide, termed VK22, derived from fowlicidin-1, a cathelicidin family of AMP from chicken oligomerizes into a predominantly tetrameric state in zwitterionic dodecylphosphocholine (DPC) micelles. An ensemble of NMR structures of VK22 determined in 200mM perdeuterated DPC, from 755 NOE constrains including 19 inter-helical NOEs, had revealed an assembly of four helices arranged in anti-parallel fashion. Hydrogen bonds, CαH―O=C types, and van der Waals interactions among the helical sub-units appear to be involved in the stabilization of the quaternary structures. The central region of the barrel shaped tetrameric bundle is non-polar with clusters of aromatic residues, whereas all the cationic residues are positioned at the termini. Paramagnetic spin labeled NMR experiments indicated that the tetrameric structure is embedded into micelles such that the non-polar region located inside the lipid acyl chains. Structure and micelle localization of a monomeric version, obtained from substitution of two Tyr residues with Ala, of the peptide is also compared. The mutated peptide VK22AA has been found be localized at the surface of the micelles. The tetrameric structure of VK22 delineates a small water pore that can be larger in the higher order oligomers. As these results provide structural insights, at atomic resolution, into the oligomeric states of a helical AMP in lipid environment, the structural details may be further utilized for the design of novel self-assembled membrane protein mimics.

Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2

Ube2g2 is an E2 enzyme which functions as part of the endoplasmic reticulum-associated degradation (ERAD) pathway responsible for identification and degradation of misfolded proteins in the endoplasmic reticulum. In tandem with a cognate E3 ligase, Ube2g2 assembles K48-linked polyubiquitin chains and then transfers them to substrate, leading ultimately to proteasomal degradation of the polyubiquitin-tagged substrate. We report here the solution structure and backbone dynamics of Ube2g2 solved by nuclear magnetic resonance spectroscopy. Although the solution structure agrees well with crystallographic structures for the E2 core, catalytically important loops (encompassing residues 95-107 and 130-135) flanking the active site cysteine are poorly defined. (15)N spin relaxation and residual dipolar coupling analysis directly demonstrates that these two loops are highly dynamic in solution. These results suggest that Ube2g2 requires one or more of its protein partners, such as cognate E3, acceptor ubiquitin substrate or thiolester-linked donor ubiquitin, to assume its catalytically relevant conformation. Within the NMR structural ensemble, interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99, supporting a possible role for His94 as a general base activated by the carboxylate side-chains of Asp98 or Asp99.

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.

PH Dependence of Stability of the 10th Human Fibronectin Type III Domain: A Computational Study

We present detailed computational studies based on electrostatic calculations to evaluate the origins of pKa values and the pH dependence of stability for the 10th type III domain of human fibronectin (FNfn10). One of our goals is to validate the calculation protocols by comparison to experimental data (Koide, A.; Jordan, M. R.; Horner, S.; Batori, V.; Koide, S. Biochemistry 2001, 40, 10326-10333). Another goal is to evaluate the sensitivity of the calculated ionization free energies and apparent pKa values on local structural fluctuations, which do not alter the structural convergence to a particular architecture, by using a complete ensemble of solution NMR structures and the NMR average minimized structure of FNfn10 (Main, A. L.; Harvey, T. S.; Baron, M.; Boyd, J.; Campbell, I. D. Cell 1992, 71, 671-678). Our calculations demonstrate that, at high ionic strength, FNfn10 is more stable at low pH compared to neutral pH, in overall agreement with experimental data. This behavior is attributed to contributions from unfavorable Coulombic interactions in a surface patch for the pairs Asp7-Glu9 and Asp7-Asp23. The unfavorable interactions are decreased at low pH, where the acidic residues become neutral, and are further decreased at high ionic strength because of increased screening by salt ions. Elimination of the unfavorable interactions in the theoretical mutants Asp7Asn (D7N) and Asp7Lys (D7K) produce higher calculated stabilities at neutral pH and any ionic strength compared to the wild-type, in agreement with the experimental data. We also discuss subtleties in the calculated apparent pKa values and ionization free energies, which are not in agreement with the experimental data. This work demonstrates that comparative electrostatic calculations can provide rapid predictions of pH-dependent properties of proteins and can be significant aids in guiding the design of proteins with tailored properties.

Solution NMR Structure of a Designed Metalloprotein and Complementary Molecular Dynamics Refinement

We report the solution NMR structure of a designed dimetal-binding protein, di-Zn(II) DFsc, along with a secondary refinement step employing molecular dynamics techniques. Calculation of the initial NMR structural ensemble by standard methods led to distortions in the metal-ligand geometries at the active site. Unrestrained molecular dynamics using a nonbonded force field for the metal shell, followed by quantum mechanical/molecular mechanical dynamics of DFsc, were used to relax local frustrations at the dimetal site that were apparent in the initial NMR structure and provide a more realistic description of the structure. The MD model is consistent with NMR restraints, and in good agreement with the structural and functional properties expected for DF proteins. This work demonstrates that NMR structures of metalloproteins can be further refined using classical and first-principles molecular dynamics methods in the presence of explicit solvent to provide otherwise unavailable insight into the geometry of the metal center.

Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL

The surface accessibility of macromolecules plays a key role in modulating molecular recognition events. RNA is a complex and dynamic molecule involved in many aspects of gene expression. However, there are few experimental methods available to measure the accessible surface of RNA. Here, we investigate the accessible surface of RNA using NMR and the small paramagnetic molecule TEMPOL. We investigated two RNAs with known structures, one that is extremely stable and one that is dynamic. For helical regions, the TEMPOL probing data correlate well with the predicted RNA surface, and the method is able to distinguish subtle variations in atom depths, such as the relative accessibility of pyrimidine versus purine aromatic carbon atoms. Dynamic motions are also detected by TEMPOL probing, and the method accurately reports a previously characterized pH-dependent conformational transition involving formation of a protonated C–A pair and base flipping. Some loop regions are observed to exhibit anomalously high accessibility, reflective of motions that are not evident within the ensemble of NMR structures. We conclude that TEMPOL probing can provide valuable insights into the surface accessibility and dynamics of RNA, and can also be used as an independent means of validating RNA structure and dynamics in solution.

Solution Structure of a Bent α-Helix,

We present the solution structure determination of a peptide with sequence Ac-WEAQAREALAKEAQARA-NH2, using NMR data. The peptide has a high population of a stable alpha-helical structure in the middle with fraying ends. In addition, our data are consistent with the presence of several other transient and interconverting conformers. The peptide sequence was designed using amino acids that have propensity for alpha-helix specificity. The presence of E-R/K (i, i + 4) ion pairs was expected to enhance the stability of the alpha-helix by introducing favorable electrostatic interactions at the side chain level, in addition to the characteristic backbone (i, i + 4) hydrogen bonds. The NMR structural ensemble demonstrates competition between E-R/K (i, i + 4) and (i, i - 1) ion pair formation, with the (i, i - 1) interactions being dominant. The relative topologies of the charged side chains demonstrate flexibility and overall compromised favorable medium/long-range electrostatic interactions. The peptide alpha-helix is bent despite the lack of an amphipathic sequence. Bending is introduced by a strong (i, i + 8) hydrophobic interaction between the side chains of N-terminal tryptophan and leucine at the middle of the peptide sequence.

Structural and Functional Characterization of Transmembrane Segment VII of the Na+/H+ Exchanger Isoform 1

The Na(+)/H(+) exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H(+) for one extracellular Na(+). It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251-273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na(+)/H(+) exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly alpha-helical, with a break in the helix at the functionally critical residues Gly(261)-Glu(262). The relative positions and orientations of the N- and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition.

Structure and Dynamics of Micelle-Associated Human Immunodeficiency Virus gp41 Fusion Domain,

The N-terminal fusion domain of the HIV-1 gp41 envelope glycoprotein is responsible for initiating the fusion of viral and cellular membranes, leading to the subsequent infection of the host cell by HIV-1. We have investigated the backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments using NMR spectroscopy and (15)N- and (15)N,(13)C,(2)H-labeled peptides. Similar (15)N-(1)H HSQC spectra were obtained in a variety of detergents, including SDS, DPC, mixed DPC/SDS, and LPPG micelles, indicating that the peptide structure is not strongly influenced by the type of detergent used. Detailed characterization was carried out in SDS micelles, where the long-term sample stability was found to be optimal. In addition to J-coupling and NOE restraints, a nearly complete set of backbone residual dipolar coupling restraints was recorded for the fusion domain-micelle complex aligned with respect to the magnetic field using a stretched polyacrylamide gel. Backbone amide (15)N spin relaxation and amide hydrogen exchange rates with the solvent were also measured. The ensemble of NMR structures reveals an uninterrupted alpha-helix for the least mobile residues (S(2) > 0.65), Ile-4 to Met-19, with transient helical character extending up to Ala-22. A 12-residue (Ile-4 to Ala-15) segment is fully shielded from solvent, with Gly-3 and Gly-16 found at micelle-solvent interfaces. Residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics relative to the residues buried in the micelle, and their mobility increases with the distance from the micelle.

Evolutionary Profiles Derived from the QR Factorization of Multiple Structural Alignments Gives an Economy of Information

We present a new algorithm, based on the multidimensional QR factorization, to remove redundancy from a multiple structural alignment by choosing representative protein structures that best preserve the phylogenetic tree topology of the homologous group. The classical QR factorization with pivoting, developed as a fast numerical solution to eigenvalue and linear least-squares problems of the form Ax=b, was designed to re-order the columns of A by increasing linear dependence. Removing the most linear dependent columns from A leads to the formation of a minimal basis set which well spans the phase space of the problem at hand. By recasting the problem of redundancy in multiple structural alignments into this framework, in which the matrix A now describes the multiple alignment, we adapted the QR factorization to produce a minimal basis set of protein structures which best spans the evolutionary (phase) space. The non-redundant and representative profiles obtained from this procedure, termed evolutionary profiles, are shown in initial results to outperform well-tested profiles in homology detection searches over a large sequence database. A measure of structural similarity between homologous proteins, Q(H), is presented. By properly accounting for the effect and presence of gaps, a phylogenetic tree computed using this metric is shown to be congruent with the maximum-likelihood sequence-based phylogeny. The results indicate that evolutionary information is indeed recoverable from the comparative analysis of protein structure alone. Applications of the QR ordering and this structural similarity metric to analyze the evolution of structure among key, universally distributed proteins involved in translation, and to the selection of representatives from an ensemble of NMR structures are also discussed.