Biomedical subjects

Ad Bax

Publications and source records attributed to Ad Bax.

At least 19 recordsLinked to original sources

PubMed2006

Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel.

A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring (15)N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone (15)N R (1), R (1rho), (15)N-{(1)H} NOE, and (15)N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsA(TM), exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, tau(c) = 38 +/- 2.5 ns, at 50 degrees C and a diffusion anisotropy, Dparallel/Dperpendicular = 0.79+/-0.05, corresponding to an aspect ratio a/b >/= 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S (2) values in the 0.2-0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps-ns time scale for the 5-residue selectivity filter, which selects K(+) ions to enter the channel.

Anisotropy↗

PubMed2006

Chemical shift tensors of protonated base carbons in helical RNA and DNA from NMR relaxation and liquid crystal measurements.

Knowledge of (13)C chemical shift anisotropy (CSA) tensors in nucleotide bases is important for interpretation of NMR relaxation data in terms of local dynamic properties of nucleic acids and for analysis of residual chemical shift anisotropy (RCSA) resulting from weak alignment. CSA tensors for protonated nucleic acid base carbons have been derived from measurements on a uniformly (13)C-enriched helical A-form RNA segment and a helical B-form DNA dodecamer at natural (13)C abundance. The magnitudes of the derived CSA principal values are tightly restricted by the magnetic field dependencies of the (13)C transverse relaxation rates, whereas the tensor orientation and asymmetry follow from quantitative measurements of interference between (13)C-{(1)H} dipolar and (13)C CSA relaxation mechanisms. Changes in the chemical shift between the isotropic and aligned states, Deltadelta, complement these measurements and permit cross-validation. The CSA tensors are determined from the experimental Deltadelta values and relaxation rates, under the assumption that the CSA tensor of any specific carbon in a given type of base is independent of the base position in either the RNA or DNA helix. However, the experimental data indicate that for pyrimidine C(6) carbons in A-form RNA the CSA magnitude is considerably larger than in B-form DNA. This result is supported by quantum chemical calculations and is attributed in part to the close proximity between intranucleotide C(6)H and O(5)' atoms in RNA. The magnitudes of the measured CSA tensors, on average, agree better with previous solid-state NMR results obtained on powdered nucleosides than with prior results from quantum chemical calculations on isolated bases, which depend rather strongly on the level of theory at which the calculations are carried out. In contrast, previously computed orientations of the chemical shift tensors agree well with the present experimental results and exhibit less dependence on the level of theory at which the computations are performed.

Anisotropy↗

PubMed2006

Pseudo-CSA restraints for NMR refinement of nucleic acid structure.

Upon alignment of oligonucleotides in a magnetic field, the downfield TROSY component of the 13C-{1H} doublet changes its resonance frequency as a result of residual 13C-1H dipolar coupling (RDC) and residual 13C chemical shift anisotropy (RCSA), and the sum of these two second rank tensors is referred to as the pseudo-CSA. The experimentally measured difference in the resonance frequency of the 13C TROSY component in the aligned and isotropic samples is referred to as residual pseudo-CSA (RPCSA), and it can be used directly as a restraint during structure calculation. Because measurement of the RPCSA involves detection of the narrow TROSY 13C doublet component, it is applicable to systems with larger rotational correlation times than RDC measurement. The method is demonstrated for structure refinement of the helical region of a 24-nt stem-loop segment or ribosomal helix-35, uniformly enriched in 13C and 15N, with RPCSA values measured at 5 and 25 degrees C. Substantial cross-validated improvements in structural accuracy are obtained upon incorporation of RPCSA restraints.

Models, Molecular↗

PubMed2006

Variable dimerization of the Ly49A natural killer cell receptor results in differential engagement of its MHC class I ligand.

Natural killer (NK) cells play a vital role in the detection and elimination of virally infected and tumor cells. The Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I molecules on target cells. Previous crystal studies revealed that the Ly49A homodimer binds one MHC molecule in an asymmetric interaction, whereas the Ly49C homodimer binds two MHC in a symmetrical fashion. Moreover, the bound receptors adopt distinctly different homodimeric forms: a "closed state" for Ly49A and an "open state" for Ly49C. Steric clashes between MHC molecules would preclude the closed Ly49A dimer from engaging two MHC in the manner of the open Ly49C dimer. To determine whether individual Ly49 receptors can undergo a conformational switch enabling them to bind MHC in different ways, we carried out a solution NMR study of unbound Ly49A, aided by dipolar coupling technology. This study reveals that, in solution, unligated Ly49A adopts a symmetric, open-state, homodimer conformation similar to that seen previously for Ly49C. Hence, Ly49A can assume both closed and open states. To address whether the Ly49A dimer can bind two MHC molecules in solution, besides the binding of one MHC observed in the crystal, we carried out analytical ultracentrifugation experiments. Velocity sedimentation demonstrates that the Ly49A dimer can engage two MHC molecules in solution, in agreement with NMR results showing that unbound Ly49A exists predominantly in the open state.

Animals↗

PubMed2006

2'-hydroxyl proton positions in helical RNA from simultaneously measured heteronuclear scalar couplings and NOEs.

The 2'-hydroxyl group in RNA plays an important structural role; it defines hydration in the minor groove, impacts thermodynamic stability of RNA, and often participates in RNA catalysis. To better study this important functional group in RNA, we describe a constant-time HMQC-IPAP-NOESY 3D NMR experiment. It simultaneously yields highly resolved 13C-separated NOEs from ribose protons to the 2'OH proton, as well as E.COSY-type measurement of JC-OH couplings, thereby permitting a quantitative study of the orientation of the 2'OH proton in RNA. The observed NOE patterns indicate that the 2'OH bonds in A-form helical RNA are primarily oriented toward the base domain, as further supported by small (1.3 +/- 0.7 Hz) 3JC1'-2'OH and relatively large (4.2 +/- 0.7 Hz) 3JC3'-2'OH couplings. The constant-time HMQC-IPAP-NOESY is suitable for measurement of interactions of rapidly exchanging protons in proteins and nucleic acids.

Nuclear Magnetic Resonance, Biomolecular↗

PubMed2006

NMR study of the tetrameric KcsA potassium channel in detergent micelles.

Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein-detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of cross- relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS-KcsA mixed micelles form an oblate spheroid with approximately 180 SDS molecules per channel. K(+) ions bind to the micelle-solubilized channel with a K(D) of 3 +/- 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel.

Amino Acid Sequence↗

PubMed2006

Carbon-13 chemical shift anisotropy in DNA bases from field dependence of solution NMR relaxation rates.

Knowledge of (13)C chemical shift anisotropy (CSA) in nucleotide bases is important for the interpretation of solution-state NMR relaxation data in terms of local dynamic properties of DNA and RNA. Accurate knowledge of the CSA becomes particularly important at high magnetic fields, prerequisite for adequate spectral resolution in larger oligonucleotides. Measurement of (13)C relaxation rates of protonated carbons in the bases of the so-called Dickerson dodecamer, d(CGCGAATTCGCG)(2), at 500 and 800 MHz (1)H frequency, together with the previously characterized structure and diffusion tensor yields CSA values for C5 in C, C6 in C and T, C8 in A and G, and C2 in A that are closest to values previously reported on the basis of solid-state FIREMAT NMR measurements, and mostly larger than values obtained by in vacuo DFT calculations. Owing to the noncollinearity of dipolar and CSA interactions, interpretation of the NMR relaxation rates is particularly sensitive to anisotropy of rotational diffusion, and use of isotropic diffusion models can result in considerable errors.

Base Sequence↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Refinement of multidomain protein structures by combination of solution small-angle X-ray scattering and NMR data.

Determination of the 3D structures of multidomain proteins by solution NMR methods presents a number of unique challenges related to their larger molecular size and the usual scarcity of constraints at the interdomain interface, often resulting in a decrease in structural accuracy. In this respect, experimental information from small-angle scattering of X-ray radiation in solution (SAXS) presents a suitable complement to the NMR data, as it provides an independent constraint on the overall molecular shape. A computational procedure is described that allows incorporation of such SAXS data into the mainstream high-resolution macromolecular structure refinement. The method is illustrated for a two-domain 177-amino-acid protein, gammaS crystallin, using an experimental SAXS data set fitted at resolutions from approximately 200 A to approximately 30 A. Inclusion of these data during structure refinement decreases the backbone coordinate root-mean-square difference between the derived model and the high-resolution crystal structure of a 54% homologous gammaB crystallin from 1.96 +/- 0.07 A to 1.31 +/- 0.04 A. Combining SAXS data with NMR restraints can be accomplished at a moderate computational expense and is expected to become useful for multidomain proteins, multimeric assemblies, and tight macromolecular complexes.

Computational Biology↗

PubMed2005

Solution structure of (gamma)S-crystallin by molecular fragment replacement NMR.

The solution structure of murine gammaS-crystallin (gammaS) has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of dipolar couplings, recorded in different alignment media, and supplemented by a small number of NOE distance restraints. gammaS consists of two topologically similar domains, arranged with an approximate twofold symmetry, and each domain shows close structural homology to closely related (approximately 50% sequence identity) domains found in other members of the gamma-crystallin family. Each domain consists of two four-strand "Greek key" beta-sheets. Although the domains are tightly anchored to one another by the hydrophobic surfaces of the two inner Greek key motifs, the N-arm, the interdomain linker and several turn regions show unexpected flexibility and disorder in solution. This may contribute entropic stabilization to the protein in solution, but may also indicate nucleation sites for unfolding or other structural transitions. The method used for solving the gammaS structure relies on the recently introduced molecular fragment replacement method, which capitalizes on the large database of protein structures previously solved by X-ray crystallography and NMR.

Animals↗

PubMed2005

Comparison of structure and dynamics of micelle-bound human alpha-synuclein and Parkinson disease variants.

Three point mutations (A30P, E46K, and A53T) as well as gene triplication genetically link the 140-residue protein alpha-synuclein (aS) to the development of Parkinson disease. Here, the structure and dynamics of micelle-bound aS(A30P) and aS(A53T) are described and compared with wild-type aS, in addition to describing the aS-micelle interaction. A53T is sensed only by directly adjacent residues and leaves the backbone structure and dynamics indistinguishable from the wild type. A30P interrupts one helix turn (Val26-Ala29) and destabilizes the preceding one. A shift in helix register following A30P disturbs the canonical succession of polar and hydrophobic residues for at least two turns. The shortened helix-N adopts a slightly higher helical content and is less bent, indicating that strain was present in the micelle-bound helix. In the vicinity of the A30P-induced perturbations, the underlying micelle environment has rearranged, but nevertheless all aS variants maintain similar interrelationships with the micelle. Moreover, aS-micelle immersion correlates well with fast and slow aS backbone dynamics, allowing a rare insight into protein-micelle interplay.

Alanine↗

PubMed2005

Measurement of ribose carbon chemical shift tensors for A-form RNA by liquid crystal NMR spectroscopy.

Incomplete motional averaging of chemical shift anisotropy upon weak alignment of nucleic acids and proteins in a magnetic field results in small changes in chemical shift. Knowledge of nucleus-specific chemical shift (CS) tensor magnitudes and orientations is necessary to take full advantage of these measurements in biomolecular structure determination. We report the determination by liquid crystal NMR of the CS tensors for all ribose carbons in A-form helical RNA, using a series of novel 3D NMR pulse sequences for accurate and resolved measurement of the ribose (13)C chemical shifts. The orientation of the riboses relative to the rhombic alignment tensor of the molecule studied, a stem-loop sequence corresponding to helix-35 of 23S rRNA, is known from an extensive set of residual dipolar couplings (RDC), previously used to refine its structure. Singular-value-decomposition fits of the chemical shift changes to this structure, or alternatively to a database of helical RNA X-ray structures, provide the CS tensor for each type of carbon. Quantum chemical calculations complement the experimental results and confirm that the most shielded tensor component lies approximately along the local carbon-oxygen bond axis in all cases and that shielding anisotropy for C3' and C4' is much larger than for C1' and C2', with C5' being intermediate.

Anisotropy↗

PubMed2005

Is the prion domain of soluble Ure2p unstructured?

The [URE3] prion is a self-propagating amyloid form of the Ure2 protein of Saccharomyces cerevisiae. Deletions in the C-terminal nitrogen regulation domain of Ure2p increase the frequency with which the N-terminal prion domain polymerizes into the prion form, suggesting that the C-terminus stabilizes the prion domain or that the structured C-terminal region sterically impairs amyloid formation. We find by in vivo two-hybrid analysis no evidence of interaction of prion domain and C-terminal domain. Furthermore, surface plasmon resonance spectrometry shows no evidence of interaction of prion domain and C-terminal domain, and cleavage at a specific site between the domains frees the two fragments. Our NMR analysis indicates that most residues of the prion domain are in fact disordered in the soluble form of Ure2p. Deleting the tether holding the C-terminal structured region to the amyloid core does not impair prion formation, arguing against steric impairment of amyloid formation. These results suggest that the N-terminal prion domain is unstructured in the soluble protein and does not have a specific interaction with the C-terminus.

Amino Acid Sequence↗

PubMed2005

Measurement of eight scalar and dipolar couplings for methine-methylene pairs in proteins and nucleic acids.

A new 3D, spin-state-selective coherence transfer NMR experiment is described that yields accurate measurements for eight scalar or dipolar couplings within a spin system composed of a methylene adjacent to a methine group. Implementations of the experiment have been optimized for proteins and for nucleic acids. The experiments are demonstrated for Cbeta-Calpha moieties of the third IgG-binding domain from Streptococcal Protein G (GB3) and for C5'-C4' groups in a 24-nt RNA oligomer. Chemical shifts of Calpha, Cbeta and Hbeta (respectively C4', C5' and H5') are dispersed in the three orthogonal dimensions, and the absence of heteronuclear decoupling leads to distinct and well-resolved E.COSY multiplet patterns. In an isotropic sample, the E.COSY displacements correspond to 1J(CalphaHalpha), 2J(CalphaHbeta2)+2J(CalphaHbeta3), 2J(CbetaHalpha), 1J(CbetaHbeta2)+1J(CbetaHbeta3), 1J(CbetaHbeta2)-2J(Hbeta2Hbeta3), 1J(CbetaHbeta3)-2J(Hbeta2Hbeta3), 3J(HalphaHbeta2) and 3J(HalphaHbeta3) for proteins, and 1J(C4'H4'), 2J(C4'H5')+2J(C4'H5"), 2J(C5'H4'), 1J(C5'H5')+1J(C5'H5"), 1J(C5'H5')-2J(H5'H5"), 1J(C5'H5")-2J(H5'H5"), 3J(H4'H5') and 3J(H4'H5") in nucleic acids. The experiment, based on relaxation-optimized spectroscopy, yields best results when applied to residues where the methine-methylene group corresponds to a reasonably isolated spin system, as applies for C, F, Y, W, D, N and H residues in proteins, or the C5'-C4' groups in nucleic acids. Splittings can be measured under either isotropic or weakly aligned conditions, yielding valuable structural information both through the 3J couplings and the one-, two- and three-bond dipolar interactions. Dipolar couplings for 10 out of 13 sidechains in GB3 are found to be in excellent agreement with its X-ray structure, whereas one residue adopts a different backbone geometry, and two residues are subject to extensive chi1 rotamer averaging. The abundance of dipolar couplings can also yield stereospecific assignments of the non-equivalent methylene protons. For the RNA oligomer, dipolar data yielded stereospecific assignments for six out of the eight C5'H2 groups in the loop region of the oligomer, in all cases confirmed by 1J(C5'H5')>1J(C5'H5"), and H5' resonating downfield of H5".

Anisotropy↗

PubMed2005

Accurate measurement of 15N-13C residual dipolar couplings in nucleic acids.

New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond 15N1/9-13C1', 15N1/9-13C6/8, and 15N1/9-13C2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ 1H-13C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H1'-C1'-N1/9 (MQ-HCN-QJ) or H6/8-C6/8-N1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and approximately 1/(2J(NC)) (attenuated spectrum). The different types of 15N-13C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time 15N evolution period and the offset of two frequency-selective 13C pulses. The methods are demonstrated for a uniformly 13C, 15N-enriched 24-nucleotide stem-loop RNA sequence, helix-35psi, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35psi sample result in an estimated precision of ca. 0.07 Hz for 1D(NC), indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of 15N1/9-13C1', 15N1/9-13C6/8, and 15N1/9-13C2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35psi, refined using independently measured observables, including 13C-1H, 13C-13C and 1H-1H dipolar couplings.

Adenine↗

PubMed2005

Weak alignment NMR: a hawk-eyed view of biomolecular structure.

Imposing a very slight deviation from the isotropic random distribution of macromolecules in solution in an NMR sample tube permits the measurement of residual internuclear dipolar couplings (RDCs). Such interactions are very sensitive functions of the time-averaged orientation of the corresponding internuclear vectors and thereby offer highly precise structural information. In recent years, advances have been made both in the technology to measure RDCs and in the computational procedures that integrate this information in the structure determination process. The exceptional precision with which RDCs can be measured under weakly aligned conditions is also starting to reveal the mostly, but not universally, subtle effects of internal protein dynamics. Importantly, RDCs potentially can reveal motions taking place on a timescale slower than rotational diffusion and analysis is uniquely sensitive to the direction of motion, not just its amplitude.

Animals↗

PubMed2005

Molecular fragment replacement approach to protein structure determination by chemical shift and dipolar homology database mining.

A novel approach is described for determining backbone structures of proteins that is based on finding fragments in the protein data bank (PDB). For each fragment in the target protein, usually chosen to be 7-10 residues in length, PDB fragments are selected that best fit to experimentally determined one-bond heteronuclear dipolar couplings and that show agreement between chemical shifts predicted for the PDB fragment and experimental values for the target fragment. These fragments are subsequently refined by simulated annealing to improve agreement with the experimental data. If the lowest-energy refined fragments form a unique structural cluster, this structure is accepted and side chains are added on the basis of a conformational database potential. The sequential backbone assembly process extends the chain by translating an accepted fragment onto it. For several small proteins, with extensive sets of dipolar couplings measured in two alignment media, a unique final structure is obtained that agrees well with structures previously solved by conventional methods. With less dipolar input data, large, oriented fragments of each protein are obtained, but their relative positioning requires either a small set of translationally restraining nuclear Overhauser enhancements (NOEs) or a protocol that optimizes burial of hydrophobic groups and pairing of beta-strands.

Computer Simulation↗

PubMed2005

Engineering DNA topology with locked nucleosides: a structural study.

DNA dodecamers modified with nucleotide building blocks based on a bicyclo[3. 1.0]hexane system that effectively locks the ribose template into an RNA-like or North (N) conformation were analyzed by various biophysical techniques including high field nuclear magnetic resonance (NMR). Replacement of either one or both of the center thymidines in the Dickerson Drew dodecamer (CGCGAAT*T*CGCG) caused a progressive shift in the bending propensity of the double helix as shown by a newly developed rapid technique that compares the residual dipolar coupling (RDC) values of the modified duplexes with those previously determined for the native DNA.

Biophysics↗