Biomedical subjects

W L Hubbell

Publications and source records attributed to W L Hubbell.

At least 55 records · Page 3Linked to original sources

PubMed1998

A conserved disulfide motif in human tear lipocalins influences ligand binding.

Structural and functional characteristics of the disulfide motif have been determined for tear lipocalins, members of a novel group of proteins that carry lipids. Amino acid sequences for two of the six isolated isoforms were assigned by a comparison of molecular mass measurements with masses calculated from the cDNA-predicted protein sequence and available N-terminal protein sequence data. A third isoform was tentatively sequence assigned using the same criteria. The most abundant isoform has a measured mass of 17 446.3 Da, consistent with residues 19-176 of the putative precursor (calculated mass 17 445.8 Da). Chemical derivatization of native and reduced/denatured protein confirmed the presence of a single intramolecular disulfide bond in the native protein. Reactivity of native, reduced, and denatured protein with 4-pyridine disulfide and dithiobis(2-nitrobenzoic acid) indicated that access to the free cysteine is markedly restricted by the intact disulfide bridge. Mass measurements of tryptic fragments identified C119 as the free cysteine and showed that the single intramolecular disulfide bond joined residues C79 and C171. Circular dichroism indicated that tear lipocalins have a predominant beta-pleated sheet structure (44%) that is essentially retained after reduction of the disulfide bond. Circular dichroism in the far-UV showed reduced molecular asymmetry and enhanced urea-induced unfolding with disulfide reduction indicative of relaxation of protein structure. Circular dichroism in the near-UV shows that the disulfide bond contributes to the asymmetry of aromatic sites. The effect of disulfide reduction on ligand binding was monitored using the intrinsic optical activity of bound retinol. The intact disulfide bond diminishes the affinity of tear lipocalins for retinol and restricts the displacement of native lipids by retinol. Disulfide reduction is accompanied by a dramatic alteration in ligand-induced conformational changes that involves aromatic residues. The disulfide bridge in tear lipocalins is important in conferring protein rigidity and influencing ligand affinity. The disulfide bond appears highly conserved so that these findings may have implications for the entire lipocalin superfamily.

Amino Acid Sequence↗

PubMed1998

Helix packing in the lactose permease determined by metal-nitroxide interaction.

The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S. A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an alpha-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.

Bacterial Proteins↗

PubMed1998

A rectangular loop-gap resonator for EPR studies of aqueous samples.

A new rectangular geometry of the loop-gap resonator for the use with a flat cell has been developed. Maxwell's equations for the resonators with two, four, six, and eight gaps have been solved assuming the existence of only the magnetic z-component. The formulas obtained were numerically solved for the electric and magnetic field distributions over the cross-sections of the resonators. The presence of a nodal plane for the electric field in the center of the resonator allows the use of a flat cell instead of a capillary for EPR measurements. Using the field distributions obtained, the quality factor and EPR signal amplitude for various shapes and gap numbers for the resonators containing a flat cell filled with water were examined numerically. This allowed finding the geometry that yields the maximum EPR signal intensity. Several X-band resonators were built in order to verify the results obtained theoretically. The experiments confirmed the ability of a novel resonant structure to accommodate a flat cell filled with an aqueous sample. It has been found that the optimum aqueous sample volume for the X-band rectangular loop-gap resonator equals 16 mm3. For a saturable aqueous sample this gives a fourfold improvement in the S/N ratio over the circular 1 mm i.d. loop-gap resonator equipped with 0.6 mm i.d. capillary.

Electron Spin Resonance Spectroscopy↗

PubMed1998

Recent advances in site-directed spin labeling of proteins.

Site-directed spin labeling of proteins is experiencing a phase of rapid technical evolution, application and evaluation. New strategies have been introduced for determining membrane protein topography, electrostatic potentials, the orientation of proteins at membrane surfaces and inter-residue distances. New applications include studies of beta strands, structure mapping using spin-spin interactions, domain motions in soluble proteins and extensive structural analysis of a number of membrane and soluble proteins.

Cell Membrane↗

PubMed1997

Structure and function in rhodopsin: topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops.

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335-T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4'-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240-V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

Amino Acid Sequence↗

PubMed1997

Structure and function in rhodopsin: rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state.

The Glu-134-Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as "molecular sensors" of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.

Animals↗

PubMed1997

Site-directed spin-labeling of transmembrane domain VII and the 4B1 antibody epitope in the lactose permease of Escherichia coli.

Functional lactose permease mutants containing single Cys residues at positions 233-255 and a biotin acceptor domain at the C terminus were solubilized in dodecyl beta-d-maltopyranoside and purified by avidin affinity chromatography. Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy (EPR). The EPR spectral line shapes and the influence of nonpolar O2 or polar potassium chromium oxalate relaxation agents on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to the relaxation agents, respectively. The results provide evidence that residues Ser233-Asn246 are within the hydrophobic core of the membrane and that Phe247 is at the lipid headgroup-solvent interface. Along with Phe247, Phe250 and Gly254 are also surface-exposed, as indicated by studies on the epitope for monoclonal antibody 4B1 [Sun, J., Wu, J., Carasco, N., and Kaback, H. R. (1996) Biochemistry 35, 990-998]. Furthermore, the nitroxide-labeled intramembrane Cys replacements exhibit variations in mobility and accessibility that are consistent with the conclusion that TM VII is an alpha-helix in contact with surrounding helices in the tertiary structure of the permease.

Amino Acid Sequence↗

PubMed1997

Arginine 302 (helix IX) in the lactose permease of Escherichia coli is in close proximity to glutamate 269 (helix VIII) as well as glutamate 325.

By using a variety of biochemical and biophysical approaches, a helix packing model for the lactose permease of Escherichia coli has been proposed in which the four residues that are irreplaceable with respect to coupling are paired--Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix XI) with Glu325 (helix X). In addition, the substrate translocation pathway is located at the interface between helices V and VIII, which is in close vicinity to the four essential residues. Based on this structural information and functional studies of mutants in the four irreplaceable residues, a molecular mechanism for energy coupling in the permease has been proposed [Kaback, H. R. (1997) Pro.c Natl. Acad. Sci. U.S.A. 94, 5539]. The principle idea of this model is that Arg302 interacts with either Glu325 or Glu269 during turnover. Evidence that Arg302 is in close proximity with Glu325 has been presented [Jung, K., Jung, H., Wu, J., Prive, G. G., & Kaback, H. R. (1993) Biochemistry 32, 12273; He, M. M., Voss, J., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 15667]; however, the proximity of Arg302 to Glu269 has not been examined. In this report, it is shown by two methods that Arg302 is also close to Glu269: (i) permease with Glu269-->His, Arg302-->His, and His322-->Phe binds Mn2+ with high affinity at pH 7.5, but not at pH 5.5; and (ii) site-directed spin-labeling of the double Cys mutant Glu269-->Cys/Arg302-->Cys exhibits spin-spin interaction with an interspin distance of about 14-16 A. In addition, the spin-spin interaction is stronger and interspin distance shorter after the permease is reconstituted into proteoliposomes. Taken as a whole, the data are consistent with the idea that Arg302 may interact with either Glu325 or Glu269 during turnover.

Amino Acid Sequence↗

PubMed1997

Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling.

T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H.R., & Matthews, B.W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 A relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X.J., Wozniak, J.A., & Matthews, B.W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.

Bacteriophage T4↗

PubMed1996

Structure and function in rhodopsin. Cysteines 65 and 316 are in proximity in a rhodopsin mutant as indicated by disulfide formation and interactions between attached spin labels.

To probe proximity relationships between different amino acids in the interhelical loops in the cytoplasmic domain of rhodopsin, we are using a general approach in which two cysteine residues are introduced at different locations. Here we report on the characteristics of one such mutant that contains the naturally occurring cysteine 316 near the cytoplasmic end of helix G and a second cysteine at position 65 (H65C), near the cytoplasmic end of helix A. The mutant protein after expression in COS-1 cells and reconstitution with 11-cis-retinal can be bound to anti-rhodopsin antibody 1D4-Sepharose at pH 6 in a form that contains the two cysteines in the free sulfhydryl form. In this form, the mutant protein reacts as expected with N-ethylmaleimide in the dark at room temperature and can be derivatized with nitroxide spin labels. However, under appropriate conditions, the mutant can be isolated with the cysteines in the disulfide form, which has been characterized by analysis of fragments produced on proteolysis with thermolysin. A study of the interactions between nitroxide spin labels attached to the two cysteine residues in the mutant protein indicates that in the dark state they are within about 10 A of each other. On illumination the distance between the spin labels increases. Collectively, the above results show that, upon folding of the mutant opsin in vivo, cysteines 65 and 316, and by inference, helices A and G, are in proximal locations and move further apart upon photoactivation.

Amino Acid Sequence↗

PubMed1996

Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin.

Conformational changes are thought to underlie the activation of heterotrimeric GTP-binding protein (G protein)-coupled receptors. Such changes in rhodopsin were explored by construction of double cysteine mutants, each containing one cysteine at the cytoplasmic end of helix C and one cysteine at various positions in the cytoplasmic end of helix F. Magnetic dipolar interactions between spin labels attached to these residues revealed their proximity, and changes in their interaction upon rhodopsin light activation suggested a rigid body movement of helices relative to one another. Disulfide cross-linking of the helices prevented activation of transducin, which suggests the importance of this movement for activation of rhodopsin.

Amino Acid Sequence↗

PubMed1996

Site-directed spin labeling demonstrates that transmembrane domain XII in the lactose permease of Escherichia coli is an alpha-helix.

Functional lactose permease mutants containing single-Cys residues at positions 387-402 [He, M. M., Sun, J., & Kaback, H. R. (1996) Biochemistry 35, 12909-12914] and a biotin acceptor domain in the middle cytoplasmic loop were solubilized in n-dodecyl-beta-D-maltopyranoside and purified by avidin affinity chromatography. Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy. Analysis of the electron paramagnetic resonance spectral line shapes and the influence of O2 on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to O2, respectively. The data show a periodic dependence of both mobility and accessibility on sequence position consistent with an alpha-helical structure. These results provide direct support for the contention that transmembrane domain XII is in an alpha-helical conformation and on the periphery of the 12-helix bundle that comprises the lactose permease molecule.

Amino Acid Sequence↗

PubMed1996

Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show position-specific effects in transducin activation.

The cytoplasmic interhelical E-F loop in rhodopsin is a part of the region that interacts with the G-protein transducin and rhodopsin kinase during signal transduction. In extending the previous work on systematic single cysteine substitutions of the amino acids in the cytoplasmic C-D loop, we have now replaced, one at a time, the amino acids Q225-I256 in the E-F loop region by cysteines. All the mutants formed the characteristic rhodopsin chromophore with 11-cis-retinal. While most of the mutants bleached normally, L226C, showed abnormal bleaching behavior. A study of the alkylation of the mutants by N-ethylmaleimide in dark showed low reactivity by some mutants, especially L226C. The rates of transducin activation (GT(alpha)-GTP gamma S complex formation) were measured for all the mutants. While these were normal for the bulk of the mutants, some (L226C, T229C, V230C, A233C, A234C, T242C, T243C, and Q244C) showed strikingly reduced transducin activation. The results suggest a specific structure in the E-F loop that interacts with transducin.

Amino Acid Sequence↗

PubMed1996

Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: a site-directed spin-labeling study.

Thirty consecutive single cysteine substitution mutants in the amino acids Q225-I256 of bovine rhodopsin have been prepared and modified with a sulfhydryl specific nitroxide reagent. This sequence includes the E-F interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with hydrophilic and hydrophobic paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra analyzed in terms of side chain mobility, both in the dark and after photoactivation. Accessibility cata shows that the rhodopsin polypeptide chain crosses an aqueous/ hydrophobic boundary in the range V227-K231 and again in the range V250-V254. In the hydrophobic segments, both the accessibility and mobility data are consistent with helical structures. In the regions of the sequence located within the aqueous phase, periodic variation in both accessibility and mobility of the spin-labeled side chains indicates that the E-F interhelical loop is largely alpha-helical, being formed by regular extensions of the E and F helices by about 1.5 and 3 turns, respectively. Judging from nitroxide mobilities, the putative extension of helix E in the aqueous phase is more dynamic than that of helix F. Changes in the electron paramagnetic resonance characteristics of the spin-labeled rhodopsin upon photoactivation indicate that chromophore isomerization results in patterns of structural changes that can be interpreted in terms of movements of helices that extend into the aqueous loop regions.

Amino Acid Sequence↗

PubMed1996

Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli.

Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label. Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII. Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228. Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs. The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A. Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A. long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226. The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII.

Amino Acid Sequence↗

PubMed1996

Organization of diphtheria toxin T domain in bilayers: a site-directed spin labeling study.

The diphtheria toxin transmembrane (T) domain was spin-labeled at consecutive residues in a helical segment, TH9. After binding of the T domain to membranes at low pH, the nitroxide side chains generated by spin labeling were measured with respect to their frequency of collision with polar and nonpolar reagents. The data showed that the helical structure of TH9 in solution is conserved, with one face exposed to water and the other to the hydrophobic interior of the bilayer. Measurement of the depth of the nitroxide side chains from the membrane surfaces revealed an incremental change of about 5 angstroms per turn, which is consistent with a transmembrane orientation of an alpha helix. These results indicate that the helix forms the lining of a transmembrane water-filled channel.

Amino Acid Sequence↗

PubMed1996

Watching proteins move using site-directed spin labeling.

Site-directed spin labeling of proteins has proven to be a practical means for determining secondary structure and its orientation; surfaces of tertiary interactions; inter-residue distances; chain topology and depth of a given side chain from the membrane/aqueous surface in membrane proteins; and local electrostatic potentials at solvent-exposed sites. Moreover, the mobility of a side chain together with its solvent-accessibility may serve to uniquely identify the topographical location of specific residues in the protein fold. Future spectral analysis should permit a quantitative estimation of the contribution of backbone flexibility to the overall side-chain dynamics. The ability to time-resolve the structural features mentioned above makes SDSL a powerful approach for exploring the evolution of structure on the millisecond time scale. We anticipate future applications to the study of protein folding both in solution and in chaperone-mediated systems.

Electron Spin Resonance Spectroscopy↗

PubMed1996

Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics.

Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure. The perturbation caused by replacement of a native residue with a nitroxide amino acid was assessed from the resulting changes in biological activity, circular dichroism, and free energy of folding. The nitroxide produced context-dependent changes in stability and activity similar to those observed for substitution with natural amino acids at the same site but had little effect on the circular dichroism spectra. At solvent-exposed sites, the structural perturbation appears to be small at the level of the backbone fold. Nitroxide side-chain mobility faithfully reflects the protein tertiary fold at all sites investigated. The primary determinants of nitroxide side-chain mobility are tertiary interactions and backbone dynamics. Tertiary interactions constrain the side-chain mobility to an extent closely correlated with the degree of interaction. At interhelical loop sites, the side chains have a high mobility, consistent with high crystallographic thermal factors. On the exposed surfaces of alpha-helices, the side-chain mobility is not restricted by interactions with nearest neighbor side chains but appears to be determined by backbone dynamics. An unexpected result is a striking difference between the mobility of residues near the C- and N-termini of helices. These results provide the foundation for another dimension of information in site-directed spin-labeling experiments that can be interpreted in terms of the protein tertiary fold, its equilibrium dynamics and time-dependent conformational changes.

Bacteriophage T4↗