PubMed Health⌕ Search

Biomedical subjects

Wayne L Hubbell

Publications and source records attributed to Wayne L Hubbell.

At least 19 recordsLinked to original sources
PubMed2006

Hydrophobic loop dynamics and actin filament stability.

It has been postulated that the hydrophobic loop of actin (residues 262-274) swings out and inserts into the opposite strand in the filament, stabilizing the filament structure. Here, we analyzed the hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop. Lateral, copper-catalyzed disulfide cross-linking of the mutant cysteine residues to the native C374 in the neighboring strand within the filament was fastest for S265C, followed by V266C, L267C, and then L269C. Site-directed spin labeling (SDSL) studies revealed that C265 lies closest to C374 within the filament, followed by C266, C267, and then C269. These results are not predicted by the Holmes extended loop model of F-actin. Furthermore, we find that disulfide cross-linking destroys L267C and L269C filaments; only small filaments are observed via electron microscopy. Conversely, phalloidin protects the L267C and L269C filaments and inhibits their disulfide cross-linking. Combined, our data indicate that, in solution, the loop resides predominantly in a "parked" position within the filament but is able to dynamically populate other conformational states which stabilize or destabilize the filament. Such states may be exploited within a cell by filament-stabilizing and -destabilizing factors.

Actin Cytoskeleton↗
PubMed2006

Structural and dynamical changes in an alpha-subunit of a heterotrimeric G protein along the activation pathway.

The Galpha subunits of heterotrimeric G proteins (Galphabetagamma) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in "switch" sequences of Galpha, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gbetagamma subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Galphai1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix alpha4, a putative effector binding region. In the inactive Galphai1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gbetagamma, motions of R1 are highly constrained, reflecting direct contact interactions at the Galphai1-Gbeta interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding alpha2/beta4 loop and smaller allosteric changes at the Galphai1-Gbetagamma interface distant from the receptor binding surface. Upon addition of GTPgammaS, the activated Galphai1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in alpha4, distant from the interaction surface, supports a role for this helix in effector binding.

Electron Spin Resonance Spectroscopy↗
PubMed2006

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe.

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 A in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 A, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA-protein complexes.

DNA↗
PubMed2006

Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins.

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP for GDP exchange on the heterotrimeric G protein alpha subunit (G alpha). Despite the crucial role of this process in cell signaling, little is known about the mechanism of G protein activation. Here we explore the structural basis for receptor-mediated GDP release using electron paramagnetic resonance spectroscopy. Binding to the activated receptor (R*) causes an apparent rigid-body movement of the alpha5 helix of G alpha that would perturb GDP binding at the beta6-alpha5 loop. This movement was not observed when a flexible loop was inserted between the alpha5 helix and the R*-binding C terminus, which uncouples R* binding from nucleotide exchange, suggesting that this movement is necessary for GDP release. These data provide the first direct observation of R*-mediated conformational changes in G proteins and define the structural basis for GDP release from G alpha.

Animals↗
PubMed2006

Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling.

Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.

Bacterial Proteins↗
PubMed2006

Conformational dynamics of loop 262-274 in G- and F-actin.

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)(2)CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)(2)CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance ( approximately 10 A) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.

Actin Cytoskeleton↗
PubMed2006

Conformational states and dynamics of rhodopsin in micelles and bilayers.

Nitroxide sensors were placed in rhodopsin at sites 140, 227, 250, and 316 to monitor the dynamics and conformation of the receptor at the cytoplasmic surface in solutions of dodecyl maltoside (DM), digitonin, and phospholipid bilayers of two compositions. The EPR spectra reveal a remarkable similarity of rhodopsin structure and the activating conformational change in DM and bilayers, the hallmark of which is an outward tilt of transmembrane helix VI. This conformational change is blocked in solutions of digitonin, although changes in optical absorbance accompany activation, showing that absorbance and structural changes are not necessarily coupled. In DM and bilayers, the receptor is apparently in equilibrium between conformational substates whose populations are modulated by activation. Despite the general similarity in the two environments, the receptor conformations have increased flexibility in DM relative to bilayers. For the activated receptor in DM and bilayers, a pH-dependent conformational equilibrium is identified that may correspond to the optically characterized MII(a)()-MII(b)() equilibrium. No specific effects of headgroup composition on receptor conformation in lipid bilayers were found.

Amino Acid Substitution↗
PubMed2006

Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin.

Arrestins regulate signaling and trafficking of G protein-coupled receptors by virtue of their preferential binding to the phosphorylated active form of the receptor. To identify sites in arrestin involved in receptor interaction, a nitroxide-containing side chain was introduced at each of 28 different positions in visual arrestin, and the dynamics of the side chain was used to monitor arrestin interaction with phosphorylated forms of its cognate receptor, rhodopsin. At physiological concentrations, visual arrestin associates with both inactive dark phosphorylated rhodopsin (P-Rh) and light-activated phosphorylated rhodopsin (P-Rh*). Residues distributed over the concave surfaces of the two arrestin domains are involved in weak interactions with both states of phosphorhodopsin, and the flexible C-terminal sequence (C-tail) of arrestin becomes dynamically disordered in both complexes. A large-scale movement of the C-tail is demonstrated by direct distance measurements using a doubly labeled arrestin with one nitroxide in the C-tail and the other in the N-domain. Despite some overlap, the molecular "footprint" of arrestin bound to P-Rh and P-Rh* is different, showing the structure of the complexes to be unique. Strong immobilizing interactions with residues in a highly flexible loop between beta-strands V and VI are only observed in complex with the activated state. This result identifies this loop as a key recognition site in the arrestin-P-Rh* complex and supports the view that flexible sequences are key elements in protein-protein interactions.

Arrestin↗
PubMed2005

Mapping of the docking of SecA onto the chaperone SecB by site-directed spin labeling: insight into the mechanism of ligand transfer during protein export.

Export of protein into the periplasm of Escherichia coli via the general secretory system is achieved by action of a ternary complex comprising the polypeptide ligand, the chaperone SecB and SecA, a peripheral component of the membrane translocon, which is itself an ATPase. The unfolded ligand is captured initially by SecB and must be transferred to SecA and subsequently through the membrane translocon into the periplasm. We have taken the first steps in the elucidation of the mechanism of this dynamic transfer by determining the interface of interaction between SecB and SecA. Site-directed spin labeling and electron paramagnetic resonance spectroscopy were combined to identify which of the residues on SecB showed changes in spectral line shape upon addition of SecA. In all, 43% of the surface of SecB was covered by the 41 positions examined. A model of docking between SecB and SecA is proposed based on the pattern of amino acid residues on SecB shown to make contacts when in complex with SecA. This model in combination with previously published biochemical data provides insight into the transfer of the unfolded polypeptide from the chaperone SecB to SecA.

Adenosine Triphosphatases↗
PubMed2005

Site-directed spin labeling studies reveal solution conformational changes in a GAAA tetraloop receptor upon Mg(2+)-dependent docking of a GAAA tetraloop.

The Mg(2+)-dependent GAAA tetraloop interaction with its 11 nucleotide receptor is one of the most frequently occurring long-range tertiary interactions in RNAs. To explore conformational changes in the receptor during tetraloop docking, nitroxide spin labels were attached at each of four uridine bases, one at a time, within an RNA molecule containing the receptor sequence. In the presence of Mg2+ and the tetraloop, the electron paramagnetic resonance (EPR) spectrum of one of the labeled bases reflected a large increase in mobility, indicating unstacking of the base upon tetraloop docking. This provides direct evidence that base unstacking is an intrinsic feature of the solution tetraloop-receptor complex formed in the presence of Mg2+. Additional evidence suggests that in solution the bound receptor conformation is similar to that observed in the crystal structure of a group I intron ribozyme domain. In Mg2+ alone, a receptor conformation with an unstacked base was not detectable, suggesting that this conformation is of higher standard state free energy than that of the free receptor. This leads to the conclusion that the extensive RNA-RNA interactions observed in the crystal structure of the tetraloop-receptor complex provide larger interaction energy than the measured apparent affinity between the tetraloop and the free receptor. This is compatible with a high specificity of the tetraloop-receptor interaction.

Base Sequence↗
PubMed2005

Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR.

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O(2) or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.

Argon↗
PubMed2005

Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR.

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.

Bacteriophage T4↗
PubMed2005

Determination of the orientation of T4 lysozyme vectorially bound to a planar-supported lipid bilayer using site-directed spin labeling.

Site-directed spin labeling is used to investigate the structure of adsorbed T4 lysozyme (T4L). A monolayer of T4L is prepared by tethering the protein selectively via a His-tag to the chelating headgroups (NTA Ni) of a planar quartz-supported lipid bilayer. This results in a vectorially oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance spectra. Similar measurements of spin-labeled lipid bilayers were used to characterize the structure and dynamics of the supports. Electron paramagnetic resonance line shape was analyzed using the stochastic Liouville equation approach developed by Freed and co-workers. The simulations reveal a conservation of the secondary and tertiary structure of T4L upon adsorption although slight conformational changes in the presence of the surface can be detected by probing tertiary contact sites. The orientation of the entire protein was deduced on the basis of an anisotropic motional model for the spin-labeled side chain. In addition, a polar order but azimuthal disorder of the molecules was assumed to fit the data. These results demonstrate the utility of site-directed spin labeling in combination with spectral simulation to study not only the secondary and tertiary structure of adsorbed proteins in monolayer coverage but also their orientation with respect to the surface.

Adsorption↗
PubMed2005

Multiquantum EPR spectroscopy of spin-labeled arrestin K267C at 35 GHz.

Three- and five-quantum absorption and dispersion multiquantum electron paramagnetic resonance spectra of a spin-labeled protein have been obtained for the first time at Q-band (35 GHz). Spectra of arrestin spin-labeled at site 267 were recorded at room temperature as a function of microwave power. The separation of irradiating microwave frequencies, Deltaf, was 10 kHz, and a newly-designed multiquantum Q-band electron paramagnetic resonance bridge was utilized, operating in a superheterodyne detection mode. The sample volume was 30 nL using a 3-loop-2-gap resonator. Most spectra were obtained at a 300 microM concentration in single, 2-min scans, but spectra were also successfully obtained at 30 microM, corresponding to one picomole of protein. Enhanced sensitivity to T(1) and T(2) was evident in the spectra, and linewidths varied considerably across the spectra. The pure absorption displays are beneficial relative to field modulation methods for spectral characterization. The presence of two states of the nitroxide spin-label with different relaxation times is evident, particularly in the dispersion spectra, which are expected to exhibit enhanced sensitivity to lineshape variation relative to absorption. Feasibility has been established for the use of this technique for site-directed spin-labeling studies of biologically relevant samples, particularly the study of protein structure and dynamics.

Absorption↗
PubMed2004

Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge.

The intramolecular interactions that stabilize the inactive conformation of rhodopsin are of primary importance in elucidating the mechanism of activation of this and other G protein-coupled receptors. In the present study, site-directed spin labeling is used to explore the role of a buried salt bridge between the protonated Schiff base at K296 in TM7 and its counterion at E113 in TM3. Spin-label sensors are placed at positions in the cytoplasmic surface of rhodopsin to monitor changes in the structure of the helix bundle caused by point mutations that disrupt the salt bridge. The single point mutations E113Q, G90D, and A292E, which were previously reported to cause constitutive activation of the apoprotein opsin, are found to cause profound movements of both TM3 and TM6 in the dark state, the latter of which is similar to that caused by light activation. The mutant M257Y, which constitutively activates opsin but does not disrupt the salt bridge, is shown to cause related but distinguishable structural changes. The double mutants E113Q/M257Y and G90D/M257Y produce strong activation of the receptor in the dark state. In the E113Q/M257Y mutant investigated with spin labeling, the movement of TM6 and other changes are exaggerated relative to either E113Q or M257Y alone. Collectively, the results provide structural evidence that the salt bridge is a key constraint maintaining the resting state of the receptor, and that the disruption of the salt bridge is the cause, rather than a consequence, of the TM6 motion that occurs upon activation.

Animals↗
PubMed2004

Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA.

In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.

DNA↗
PubMed2004

Structure and dynamics of a helical hairpin that mediates calcium-dependent membrane binding of annexin B12.

A wealth of high-resolution structural data has accumulated for soluble annexins, but only limited information is available for the biologically important membrane-bound proteins. To investigate the structural and dynamic changes that occur upon membrane binding, we analyzed the electron paramagnetic resonance (EPR) mobility and accessibility parameters of a continuous 30-residue nitroxide scan encompassing helices D and E in repeat 2 of annexin B12 (residues 134-163) while the protein was bound to phospholipid vesicles in the presence of Ca(2+). A comparison of these data to those from a previously published study of the protein in solution (Isas, J. M., Langen, R., Haigler, H. T., and Hubbell, W. L. (2002) Biochemistry 41, 1464-1473) showed that the overall backbone fold for the scanned region did not change upon membrane binding. However, side-chains in the loop between the D and E helices were highly dynamic in solution but became essentially frozen in the EPR time scale upon binding to membranes. Accessibility measurements clearly established that side-chains in this loop were exposed to the hydrophobic core of the bilayer and provide the first evidence that a D-E loop directly participates in the Ca(2+)-dependent binding of annexins to membranes. Other localized changes showed that the D-helix became much less dynamic after membrane binding and identified quaternary contact sites in the membrane-bound homo-trimer. Finally, immobilization of the D-E loop upon contact with phospholipid suggests that the bilayer, which is normally very mobile on the EPR time scale, is immobilized in the head-group region by the annexin B12. This suggests that annexin B12 alters membrane structure in a manner that may be biologically significant.

Annexins↗