Search PubMed⌕ Search

Biomedical subjects

W L Hubbell

Publications and source records attributed to W L Hubbell.

At least 19 recordsLinked to original sources
PubMed2003

Structure and dynamics of annexin 12 bound to a planar lipid bilayer.

Site directed spin labeling is used to investigate the protein annexin 12 absorbed on a single planar phospholipid bilayer of approximately 2-3 cm(2). Electron paramagnetic resonance spectra of nitroxide side chain at several topological sites reveal a conserved tertiary fold of the protein in the absorbed state, in agreement with earlier diffraction results. The angular dependent spectra of the two-dimensional microcrystals are shown to provide information on the degree of ordering of spin labels in a alpha-helix and in turn on the orientation of the alpha-helix with respect to the surface.

Annexins↗
PubMed2001

Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations.

Magnetic dipolar interactions between pairs of solvent-exposed nitroxide side chains separated by approximately one to four turns along an alpha-helix in T4 lysozyme are investigated. The interactions are analyzed both in frozen solution (rigid lattice conditions) and at room temperature as a function of solvent viscosity. At room temperature, a novel side chain with hindered internal motion is used, along with a more commonly employed nitroxide side chain. The results suggest that methods developed for rigid lattice conditions can be used to analyze dipolar interactions between nitroxides even in the presence of motion of the individual spins, provided the rotational correlation time of the interspin vector is sufficiently long. The distribution of distances observed for the various spin pairs is consistent with rotameric equilibria in the nitroxide side chain, as observed in crystal structures. The existence of such distance distributions places important constraints on the interpretation of internitroxide distances in terms of protein structure and structural changes.

Amino Acids↗
PubMed2001

Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 316 in helix 8 and residues in the sequence 60-75, covering the cytoplasmic end of helices TM1 and TM2 and their connection loop CL1.

Double-spin-labeled mutants of rhodopsin were prepared containing a nitroxide side chain at position 316 in the cytoplasmic surface helix H8, and a second nitroxide in the sequence of residues 60-75, which includes the cytoplasmic loop CL1 and cytoplasmic ends of helices TM1 and TM2. Magnetic dipole-dipole interactions between the spins were analyzed to provide interspin distance distributions in both the dark and photoactivated states of rhodopsin. In the dark state in solutions of dodecyl maltoside, the interspin distances are found to be consistent with structural models of the nitroxide side chain and rhodopsin, both derived from crystallography. Photoactivation of rhodopsin shows a pattern of increases in internitroxide distance between the reference, position 316 in H8, and residues in CL1 and TM2 that suggests an outward displacement of TM2 relative to H8 by approximately 3 A.

Amino Acid Sequence↗
PubMed2001

Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 65 in helix TM1 and residues in the sequence 306-319 at the cytoplasmic end of helix TM7 and in helix H8.

Spin-labeled double mutants of rhodopsin were produced containing a reference nitroxide at position 65, at the cytoplasmic termination of helix TM1, and a second nitroxide in the sequence of residues 306-319, which includes the cytoplasmic termination of helix TM7 and nearly the entire surface helix H8. Magnetic dipole-dipole interactions between the spins are analyzed to provide interspin distance distributions in both the dark and photoactivated states of rhodopsin. The distributions, apparently resulting from the conformational flexibility of the side chains, are found to be consistent with the structural model of rhodopsin in the dark state derived from crystallography. Photoactivation of the receptor triggers an increase in distance between residues in TM7, but not those in H8, relative to the reference at position 65 in TM1. The simplest interpretation of the result is a movement of the cytoplasmic portion of TM7 away from TM1 by 2-4 A.

Amino Acid Sequence↗
PubMed2001

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1.

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is presumed to be the key to the restriction of binding of transducin and rhodopsin kinase to rhodopsin. Upon light-activation, this tertiary structure undergoes a conformational change to form a new structure, which is recognized by the above proteins and signal transduction is initiated. In this and the following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-12485], we probe the dark state cytoplasmic domain structure in rhodopsin by investigating proximity between amino acids in different regions of the cytoplasmic face. The approach uses engineered pairs of cysteines at predetermined positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original amino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic sequence connecting helices I-II, disulfide bond formation was studied under strictly defined conditions and plotted as a function of the position of the variable cysteines. An absolute maximum was observed for position 65 with two additional relative maxima for cysteines at positions 61 and 68. The observed disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small but significant motions are required for productive disulfide bond formation. During these motions, secondary structure elements are retained as indicated by the lack of disulfide bond formation in cysteines that do not face toward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.

Amino Acid Sequence↗
PubMed2001

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between cysteine pairs engineered in cytoplasmic loops 1, 3, and 4.

To probe proximities between amino acids in the cytoplasmic domain by using mutants containing engineered cysteine pairs, three sets of rhodopsin mutants have been prepared. In the first two sets, a cysteine was placed, one at a time, at positions 311-314 in helix VIII, while the second cysteine was fixed at position 246 (set I) and at position 250 (set II) at the cytoplasmic end of helix VI. In the third set, one cysteine was fixed at position 65 while the second cysteine was varied between amino acid positions 306 and 321 located at the cytoplasmic end of helix VII and throughout in helix VIII. Rapid disulfide bond formation in the dark was found between the cysteine pairs in mutants A246C/Q312C,A246C/K311C and in mutants H65C/C316, H65C/315C and H65C/312C. Disulfide bond formation at much lower rates was found in mutants A246C/F313C, V250C/Q312C, H65C/N310C, H65C/K311C, H65C/F313C, and H65C/R314C; the remaining mutants showed no significant disulfide bond formation. Comparisons of the results from disulfide bond formation in solution with the distances observed in the rhodopsin crystal structure showed that the rates of disulfide bond formation in most cases were consistent with the amino acid proximities as revealed in crystal structure. However, deviations were also found, in particular, in the set containing fixed cysteine at position Cys246 and cysteines at positions 311-314. The results implicate significant effects of structural dynamics on disulfide bond formation in solution.

Amino Acid Sequence↗
PubMed2001

Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.

Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of the N-terminal domain (positions 18 and 29) and two Cys residues located within the C-terminal lobe (positions 106 and 125) were employed to detect conformational changes upon Ca(2+) binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environmentally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate, a spin label reagent, were studied using fluorescence and EPR spectroscopy, respectively. Only minor structural changes around Cys(18), Cys(29), Cys(106), and Cys(125) were observed as a function of Ca(2+) concentration. No Ca(2+)-dependent oligomerization of GCAP1 was observed at physiologically relevant Ca(2+) concentrations, in contrast to the observation reported by others for GCAP2. Based on these results and previous studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca(2+) dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its partner, guanylyl cyclase, from an inactive (or low activity) to an active conformation.

Amino Acid Motifs↗
PubMed2001

Identification of a subunit interface in transthyretin amyloid fibrils: evidence for self-assembly from oligomeric building blocks.

Amyloid and prion diseases appear to stem from the conversion of normally folded proteins into insoluble, fiber-like assemblies. Despite numerous structural studies, a detailed molecular characterization of amyloid fibrils remains elusive. In particular, models of amyloid fibrils proposed thus far have not adequately defined the constituent protein subunit interactions. To further our understanding of amyloid structure, we employed thiol-specific cross-linking and site-directed spin labeling to identify specific protein-protein associations in transthyretin (TTR) amyloid fibrils. We find that certain cysteine mutants of TTR, when dimerized by chemical cross-linkers, still form fibers under typical in vitro fibrillogenic conditions. In addition, site-directed spin labeling of many residues at the natural dimer interface reveals that their spatial proximity is preserved in the fibrillar state even in the absence of cross-linking constraints. Here, we present the first view of a subunit interface in TTR fibers and show that it is very similar to one of the natural dimeric interchain associations evident in the structure of soluble TTR. The results clarify varied models of amyloidogenesis by demonstrating that transthyretin amyloid fibrils may assemble from oligomeric protein building blocks rather than structurally rearranged monomers.

Amyloid↗
PubMed2001

Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study.

The GNRA (N: any nucleotide; R: purine) tetraloop/receptor interaction is believed to be one of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated tetraloop/receptor complex has not been identified in solution. In the present work, site-directed spin labeling is applied to detect tetraloop/receptor complex formation and estimate the free energy of interaction. For this purpose, the GAAA tetraloop/receptor interaction was chosen as a model system. A method was developed to place nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA tetraloop. Formation of the tetraloop/receptor complex was monitored through changes in the rotational correlation time of the tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA tetraloop forms a complex with an RNA containing the 11-nucleotide GAAA tetraloop receptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 mM MgCl2, Kd = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategy presented here should have broad utility in quantifying weak interactions that would otherwise be undetectable, for both nucleic acids and nucleic acid-protein complexes.

Animals↗
PubMed2001

Molecular motion of spin labeled side chains in alpha-helices: analysis by variation of side chain structure.

Two single cysteine substitution mutants at helix surface sites in T4 lysozyme (D72C and V131C) have been modified with a series of nitroxide methanethiosulfonate reagents to investigate the structural and dynamical origins of their electron paramagnetic resonance spectra. The novel reagents include 4-substituted derivatives of either the pyrroline or pyrrolidine series of nitroxides. The spectral line shapes were analyzed as a function of side chain structure and temperature using a simulation method with a single order parameter and diffusion rates about three orthogonal axes as parameters. Taken together, the results provide strong support for an anisotropic motional model of the side chain, which was previously proposed from qualitative features of the spectra and crystal structures of spin labeled T4 lysozyme. Site-specific differences in apparent order parameter are interpreted in terms of backbone dynamics modes with characteristic correlation times in the nanosecond or faster time scale. The saturated 4-substituted pyrrolidine nitroxides are shown to be a suitable template for novel "functionalized" side chains designed to mimic salient features of the native side chains they replace.

Amino Acid Substitution↗
PubMed2001

Helix packing in the lactose permease of Escherichia coli: distances between site-directed nitroxides and a lanthanide.

By exploiting substrate protection of Cys148 in lactose permease, a methanethiosulfonate nitroxide spin-label was directed specifically to one of two Cys residues in a double-Cys mutant, followed by labeling of Cys148 with a thiol-reactive chelator that binds Gd(III) quantitatively. Distances between bound Gd(III) and the nitroxide spin-label were then studied by electron paramagnetic resonance. The results demonstrate that the Gd(III)-induced relaxation effects on nitroxides at positions 228, 226 (helix VII), and 275 (helix VIII) agree qualitatively with results obtained by studying spin-spin interactions [Wu, J., Voss, J., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 10123-10127]. Thus, a nitroxide attached to position 228 (helix VII) is closest to the lanthanide at position 148 (helix V), a nitroxide at position 275 (helix VIII) is further away, and the distance between positions 226 (helix VII) and 148 is too long to measure. However, the Gd(III)-spin-label distances are significantly longer than those estimated from nitroxide-nitroxide interactions between the same pairs due to the nature of the chelator. Although the results provide strong confirmation for the contention that helix V lies close to both helices VII and VIII in the tertiary structure of lactose permease, other methods for binding rare earth metals are discussed which do not involve the use of bulky chelators with long linkers.

Binding Sites↗
PubMed2000

Binding of spin-labeled galactosides to the lactose permease of Escherichia coli.

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.

Alkylating Agents↗
PubMed2000

Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure.

High resolution (1.43-1.8 A) crystal structures and the corresponding electron paramagnetic resonance (EPR) spectra were determined for T4 lysozyme derivatives with a disulfide-linked nitroxide side chain [-CH(2)-S-S-CH(2)-(3-[2,2,5,5-tetramethyl pyrroline-1-oxyl]) identical with R1] substituted at solvent-exposed helix surface sites (Lys65, Arg80, Arg119) or a tertiary contact site (Val75). In each case, electron density is clearly resolved for the disulfide group, revealing distinct rotamers of the side chain, defined by the dihedral angles X(1) and X(2). The electron density associated with the nitroxide ring in the different mutants is inversely correlated with its mobility determined from the EPR spectrum. Residue 80R1 assumes a single g(+)()g(+)() conformation (Chi(1) = 286, X(2) = 294). Residue 119R1 has two EPR spectral components, apparently corresponding to two rotamers, one similar to that for 80R1 and the other in a tg(-)() conformation (Chi(1) = 175, X(2) = 54). The latter state is apparently stabilized by interaction of the disulfide with a Gln at i + 4, a situation also observed at 65R1. R1 residues at helix surface site 65 and tertiary contact site 75 make intra- as well as intermolecular contacts in the crystal and serve to identify the kind of molecular interactions possible for the R1 side chain. A single conformation of the entire 75R1 side chain is stabilized by a variety of interactions with the nitroxide ring, including hydrophobic contacts and two unconventional C-H.O hydrogen bonds, one in which the nitroxide acts as a donor (with tyrosine) and the other in which it acts as an acceptor (with phenylalanine). The interactions revealed in these structures provide an important link between the dynamics of the R1 side chain, reflected in the EPR spectrum, and local protein structure. A library of such interactions will provide a basis for the quantitative interpretation of EPR spectra in terms of protein structure and dynamics.

Bacteriophage T4↗
PubMed2000

Time-resolved site-directed spin-labeling studies of bacteriorhodopsin: loop-specific conformational changes in M.

A spin-label at site 101 in the C-D loop of bacteriorhodopsin was previously found to detect a conformational change during the M --> N transition [Steinhoff, H. -J., Mollaaghababa, R., Altenbach, C., Hideg, K., Krebs, M. P., Khorana, H. G., and Hubbell, W. L. (1994) Science 266, 105-107]. We have extended these time-resolved electron paramagnetic resonance studies in purple membranes by analyzing conformational changes detected by a spin-label at another site in the C-D loop (103), and at sites in the A-B loop (35), the D-E loop (130), and the E-F loop (160). In addition, we have investigated the motion detected by a spin-label at site 101 in a D96A mutant background that has a prolonged M intermediate. We find that among the examined sites, only spin-labels in the C-D loop detect a significant change in the local environment after the rise of M. Although the D96A mutation dramatically prolongs the lifetime of the M intermediate, it does not perturb either the structure of bacteriorhodopsin or the nature of the light-activated conformational change detected by a spin-label at site 101. In this mutant, a conformational change is detected during the lifetime of M, when no change in the 410 nm absorbance is observed. These results provide direct structural evidence for the heterogeneity of the M population in real time, and demonstrate that the motion detected at site 101 occurs in M, prior to Schiff base reprotonation.

Alanine↗
PubMed2000

Identifying conformational changes with site-directed spin labeling.

Site-direct spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structural changes in proteins. This review provides examples that illustrate strategies for interpreting the data in terms of specific rearrangements in secondary and tertiary structure. The changes in the mobility and solvent accessibility of the spin label side chains, and in the distances between spin labels, report (i) rigid body motions of alpha-helices and beta-strands (ii) relative movements of domains and (iii) changes in secondary structure. Such events can be monitored in the millisecond time-scale, making it possible to follow structural changes during function. There is no upper limit to the size of proteins that can be investigated, and only 50-100 picomoles of protein are required. These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems.

Animals↗
PubMed2000

Protein global fold determination using site-directed spin and isotope labeling.

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 A were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 A from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.

Amino Acid Substitution↗
PubMed1999

Nitroxide scanning electron paramagnetic resonance of helices IV and V and the intervening loop in the lactose permease of Escherichia coli.

Glu126 and Arg144 in helices IV and V, respectively, in the lactose permease of Escherichia coli, which play an indispensable role in substrate binding, are charge-paired and in close proximity [Venkatesan, P., Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807; Zhao, M., Zen, K.-C., et al. (1999) Biochemistry 38, 7407-7412]. Since hydropathy plots indicate that these residues are at the membrane-water interface at the cytoplasmic surface of the membrane, site-directed nitroxide scanning electron paramagnetic resonance (EPR) has been carried out on this region of the permease. Thirty-one single-Cys permease mutants were spin-labeled and examined by conventional and power saturation EPR. The motional freedom of the side chains, as well as accessibility to O(2) or potassium chromium oxalate (CrOx), indicates that the loop between helices IV and V (loop IV/V) is considerably smaller than predicted by hydropathy plots, extending only from about Val132 to Phe138 and that Glu126 and Arg144 are probably within the membrane. Although ligand binding has no effect on the mobility of the labeled side chains, a marked increase in CrOx and O(2) accessibility is observed at position 137, as well as significant changes in accessibility to CrOx on one face of helix V. It is concluded that ligand binding induces a conformational change in the vicinity of the binding site, resulting in increased accessibility of position 137 in loop IV/V to solvent.

Amino Acid Sequence↗