Biomedical subjects

W L Hubbell

Publications and source records attributed to W L Hubbell.

At least 73 records · Page 4Linked to original sources

PubMed1996

Calculation of electron paramagnetic resonance spectra from Brownian dynamics trajectories: application to nitroxide side chains in proteins.

We present a method to simulate electron paramagnetic resonance spectra of spin-labeled proteins that explicitly includes the protein structure in the vicinity of the attached spin label. The method is applied to a spin-labeled polyleucine alpha-helix trimer. From short (6 ns) stochastic dynamics simulations of this trimer, an effective potential energy function is calculated. Interaction with secondary and tertiary structures determine the reorientational motion of the spin label side chains. After reduction to a single particle problem, long stochastic dynamic trajectories (700 ns) of the spin label side-chain reorientation are calculated from which the Lamor frequency trajectory and subsequently the electron paramagnetic resonance spectrum is determined. The simulated spectra agree well with experimental electron paramagnetic resonance spectra of bacteriorhodopsin mutants with spin labels in similar secondary and tertiary environments as in the polyleucine.

Cyclic N-Oxides↗

PubMed1996

Expression of bcl-2 inhibits cellular radical generation.

Bcl-2 expression in neural cells has been shown to inhibit apoptotic death in association with a decrease in reactive oxygen species. We present the results of a study that used electron spin resonance (ESR) measurements to evaluate the level of hydroxyl radical production in bcl-2 expressing GT1-7 cells and control cells. Incubation of cell monolayers with the spin trap N-t-alpha-phenylnitrone (PBN), and measurements of the hydroxyl radical production at different timepoints, revealed a higher radical production in control cells than in bcl-2 expressing cells, even in the absence of insult. The ESR signal was suppressed by addition of ethanol, indicating that the trapped radical was indeed hydroxyl radical. The mechanism by which the expression of bcl-2 leads to a decrease in cellular production of hydroxyl radical is unknown.

Animals↗

PubMed1995

A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme.

The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 --> His/Gln-69 --> His) of three mutants, each containing a single nitroxide-labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II)-induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 A using Redfield theory, as first suggested by Leigh [Leigh, J.S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease.

Bacteriophage T4↗

PubMed1995

Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Amino Acid Sequence↗

PubMed1995

Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 1. Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358).

The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI). Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding. The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237 --> His/Lys358 --> His and Asp240 --> His/Lys319 --> His exhibit significant transport activity while the single-His mutants Lys319 --> His and Lys358 --> His are inactive. (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240 --> His/Lys319 --> His and Asp237 --> His/Lys358 --> His are inactive; however, the single-His mutant Lys319 --> His exhibits significant activity. (iii) The single-His mutant Asp237 --> His or ASP240 --> His is inactive at all pH values tested. In addition, a pH titration of Asp237 --> His/Lys358 --> His permease activity exhibits a midpoint at about 6.2. Finally, the purified mutant proteins Asp237 --> His/Lys358 --> His and Asp240 --> His/Lys319 --> His were assayed for Mn2+ binding by electron paramagnetic resonance spectroscopy. Asp237 --> His/Lys358 --> His permease binds Mn2+ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 2. Proximity of helix IX (Arg302) with helix X (His322 and Glu325).

Engineering divalent metal-binding sites into the lactose permease of Escherichia coli by introducing bis-His residues has been utilized to confirm the proximity of helices VIII (Glu269 --> His) and X (His322) [Jung, K., Voss, J., He, M., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 6272] and helices VII (Asp237 --> His) and XI (Lys358 --> His) [He, M. M., Voss, J., Hubbell, W. L., & Kaback, H.R. (1995) Biochemistry 34, 00000--00000]. In this paper, the approach is used to confirm and extend the relationship between helices IX (Arg302) and X (His322 and Glu325) [Jung, K., Jung, H., Wu, J., Prive, G. G., l& Kaback, H. R. (1993) Biochemistry 32, 12273]. Thus, mutants Arg302 --> His, Glu325 --> His, and Arg302 --> His/Glu325 --> His were constructed, and Mn2+ binding was assayed by electron paramagnetic resonance. Mutant Arg302 --> His binds Mn2+ with a KD of about 24 microM and a stoichiometry approximating unity in all likelihood because the His residue at position 302 forms a metal-binding site in conjunction with the native His residue at position 322. Mutant Arg302 --> His/Glu325 --> His also binds Mn2+ with a 1:1 stoichiometry, but the KD is decreased to about 13 microM. The results suggest that Arg302 is sufficiently close to both Glu325 and His322 to form a tridentate metal-binding site in mutant Arg302 --> His/Glu325 --> His. In contrast, replacement of Glu325 with His in permease with a native His residue at position 322 does not lead to Mn2+ binding. The results provide strong support for the helix packing model proposed.

Amino Acid Sequence↗

PubMed1995

Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacY-phoA fusion analysis and site-directed spectroscopy.

The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane [Calamia, J., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937-4941]. We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively. For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI. A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Try228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices. Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237 --> Cys or Lys358 --> Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains. However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane. The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.

Alkaline Phosphatase↗

PubMed1995

Mapping light-dependent structural changes in the cytoplasmic loop connecting helices C and D in rhodopsin: a site-directed spin labeling study.

All 20 single cysteine substitution mutants in the sequence Y136-M155 of bovine rhodopsin have been prepared and modified with a sulfhydryl-specific nitroxide reagent. This sequence contains the C-D interhelical loop, a transducin interaction site. The accessibilities of the attached nitroxides to collisions with paramagnetic probes in solution were determined, and the electron paramagnetic resonance spectra were analyzed, both in the dark and after photoexcitation. Accessibility data show that the rhodopsin polypeptide crosses an aqueous/hydrophobic boundary near V138 and H152. The nitroxide mobilities inferred from the spectra are consistent with a model where the C helix extends to at least residue C140, with much of the helix surface in contact with protein rather than lipid near the cytoplasmic surface of the membrane. Upon photoexcitation, electron paramagnetic resonance spectral changes are observed at sites on the putative C helix surface that are in contact with the protein and at specific sites in the C-D interhelical loop. A simple interpretation of these results is that photoexcitation involves a rigid body movement of the C helix relative to the others in the helix bundle.

Amino Acid Sequence↗

PubMed1995

Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli.

Site-directed excimer fluorescence indicates that Glu269 (helix VIII) and His322 (helix X) in the lactose permease of Escherichia coli lie in close proximity [Jung, K., Jung, H., Wu, J., Privé, G.G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. In this study, Glu269 was replaced with His in wild-type permease, leading to the presence of bis-His residues between helices VIII and X. Wild-type and Glu269-->His permease containing a biotin acceptor domain were purified by monomeric avidin affinity chromatography, and binding of Mn2+ was studied by electron paramagnetic resonance (EPR) spectroscopy. The amplitude of the Mn2+ EPR spectrum is reduced by the Glu269-->His mutant, while no change is observed in the presence of wild-type permease. The Glu269-->His mutant contains a single binding site for Mn2+ with a KD of about 43 microM, and Mn2+ binding is pH dependent with no binding at pH 5.0, stoichiometric binding at pH 7.5, and a midpoint at about pH 6.3. The results confirm the conclusion that helices VIII and X are closely opposed in the tertiary structure of lac permease and provide a novel approach for studying helix proximity, as well as solvent accessibility, in polytopic membrane proteins.

Binding Sites↗

PubMed1995

Interaction of the isolated transmembrane domain of diphtheria toxin with membranes.

Insertion of diphtheria toxin's T (transmembrane) domain into the endosomal membrane under acidic conditions is known to promote translocation of its catalytic domain across the membrane and into the cytosol. The T domain, a cysteine-free bundle of alpha-helices, was expressed as a discrete protein in Escherichia coli and purified. The isolated domain was stable and largely monomeric at pH 8.0. Like the holotoxin it bound the hydrophobic fluorophore, 2-p-toluidinylnaphthalene 6-sulfonate, upon acidification, but the transition pH was higher than with the holotoxin (pH 5.6 vs 5.1) and broader, reflecting the absence of interdomain interactions. The domain also permeabilized large unilamellar vesicles under acidic conditions, as demonstrated by release of entrapped solutes. Mutant forms of T domain, each with a single residue replaced by cysteine, were derivatized with a thiol-reactive nitroxide-containing spin label and analyzed by electron paramagnetic resonance (EPR). EPR spectra and solvent accessibilities of the labels at pH 8.0 were consistent with the environments predicted from the toxin's crystallographic structure. Acidification in the presence of large unilamellar vesicles caused a nitroxide label at position 332 on helix TH8 to move from a buried site in the water soluble state to a lipid-exposed surface site at a depth of approximately 15 A within the bilayer. This is consistent with the concept that the TH8-TH9 helix pair inserts into the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1995

Interaction of alpha-crystallin with spin-labeled peptides.

alpha-Crystallin is a major protein of the vertebrate lens once thought to be highly specialized for conferring transparency. However, recent work has revealed a wide tissue distribution and a sequence homology to small heat shock proteins, suggesting a more general role for the protein. Like other molecular chaperons, alpha-crystallin is known to bind to unfolded proteins and suppress nonspecific aggregation in vitro. In the present work, spin-labeled derivatives of the insulin B chain and melittin were used to investigate the state of these proteins bound to alpha-crystallin. Insulin was selected since unfolding can be triggered by reduction of the interchain disulfide bonds, a treatment that does not affect alpha-crystallin. Upon reduction of insulin, the separated B chains aggregate. In the presence of alpha-crystallin, the B chains bind to alpha-crystallin and aggregation is suppressed. Melittin, a 26 amino acid peptide from bee venom, was selected for study since it is a random coil under physiological conditions, and its interaction with alpha-crystallin can be directly studied. EPR analysis of the spin-labeled peptides shows that the nitroxide side chains are immobilized in a polar environment on alpha-crystallin and that they are separated by 25 A or more in the complex, indicating that the bound proteins are not clustered. The bound B chains of insulin are not in a fully extended conformation, and melittin does not appear to bind to a hydrophobic surface in alpha-crystallin as an amphipathic helix, as it does to membranes and some other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallins↗

PubMed1995

Tear lipocalins bind a broad array of lipid ligands.

To identify the native ligands of tear lipocalins, tear proteins were separated by size exclusion chromatography and the lipid content in the major protein fractions identified. Lipids extracted from native tears and purified tear lipocalins comigrated with fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol on thin layer chromatograms. Abundant stearic and palmitic acids as well as cholesterol, and lesser amounts of lauric acid were specifically identified in extracts of purified lipocalins by gas chromatography-mass spectroscopy. A preliminary study of the ligand-protein interaction was carried out using nitroxide spin-labeled lipids.

Carrier Proteins↗

PubMed1994

Time-resolved detection of structural changes during the photocycle of spin-labeled bacteriorhodopsin.

Bacteriorhodopsin was selectively spin labeled at residues 72, 101, or 105 after replacement of the native amino acids by cysteine. Only the electron paramagnetic resonance spectrum of the label at 101 was time-dependent during the photocycle. The spectral change rose with the decay of the M intermediate and fell with recovery of the ground state. The transient signal is interpreted as the result of movement in the C-D or E-F interhelical loop, or in both, coincident with protonation changes at the key aspartate 96 residue. These results link the optically characterized intermediates with localized conformational changes in bacteriorhodopsin during the photocycle.

Bacteriorhodopsins↗

PubMed1994

Site-specific incorporation of biophysical probes into proteins.

Biophysical probes which can detect structural changes in proteins and the interaction of proteins with other macromolecules are important tools in studying protein function. Many difficulties remain, however, in introducing probes into proteins site-specifically. Here we report the successful site-specific incorporation of a spin-labeled, a fluorescent, and a photoactivatible amino acid into a variety of surface and internal sites in bacteriophage T4 lysozyme by using unnatural amino acid mutagenesis. In addition, we report the purification and spectral characterization of T4 lysozyme mutants containing the spin-labeled amino acid and the fluorescent amino acid. The ability to incorporate these probes site-specifically allows for novel studies of protein structure and dynamics. Moreover, this work demonstrates that the Escherichia coli protein biosynthetic machinery can tolerate unnatural amino acids with little resemblance to the natural amino acids.

Bacteriophage T4↗

PubMed1994

A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin.

Ten mutants of bacteriorhodopsin, each containing a single cysteine residue regularly spaced along helix D and facing the lipid bilayer, were derivatized with a nitroxide spin label. Collision rates of the nitroxide with apolar oxygen increased with distance from the membrane/solution interface. Collision rates with polar metal ion complexes decreased over the same distance. Although the collision rates depend on steric constraints imposed by the local protein structure and on the depth in the membrane, the ratio of the collision rate of oxygen to those of a polar metal ion complex is independent of structural features of the protein. The logarithm of the ratio is a linear function of depth within the membrane. Calibration of this ratio parameter with spin-labeled phospholipids allows localization of the individual nitroxides, and hence the bacteriorhodopsin molecule, relative to the plane of the phosphate groups of the bilayer. The spacing between residues is consistent with the pitch of an alpha-helix. These results provide a general strategy for determining the immersion depth of nitroxides in bilayers.

Bacteriorhodopsins↗

PubMed1993

Photoactivated conformational changes in rhodopsin: a time-resolved spin label study.

Rhodopsin has been selectively spin-labeled near the cytoplasmic termini of helices C and G. Photoactivation with a light flash induces an electron paramagnetic resonance spectral change in the millisecond time domain, coincident with the appearance of the active metarhodopsin II intermediate. The spectral change is consistent with a small movement near the cytoplasmic termination of the C helix and reverses upon formation of the MIII state. These results provide an important link between the optical changes associated with the retinal chromophore and protein conformational states.

Amino Acid Sequence↗

PubMed1993

Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.

Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65, 140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.

Amino Acid Sequence↗

PubMed1993

Voltage activation of reconstituted sodium channels: use of bacteriorhodopsin as a light-driven current source.

We present a general method for the development and control of transmembrane potentials (delta psi) in reconstituted vesicles. The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium halobium is the current source in the system, and the intensity of light controls the magnitude of delta psi at any given time. Transmembrane potentials were determined from the equilibrium distribution of hydrophobic ions, which was monitored by using either electron paramagnetic resonance spectroscopy and spin-labeled phosphonium ions or a tetraphenylphosphonium-selective electrode. A bias or holding potential was generated by using gradients of anions of limited permeability in the presence of an impermeable cation. Using n-methylglucamine or the polymer poly(ethylene imide) as the impermeable cation, the anions NO3- and SCN- were most effective in producing large (> -60 mV) negative diffusion potentials in egg phosphatidylcholine vesicles. In the presence of a NO(3-)-based negative holding potential (approx. -65 mV), bR is capable of depolarizing the membrane to at least the 0-mV level within a few seconds. More rapid depolarizations can be achieved by the application of a brief intense illumination preceding the preset illumination level (supercharging). The technique was successfully used to activate for the first time a population of unmodified voltage-dependent sodium channels purified from eel electroplax.

Bacteriorhodopsins↗