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.

Colicin E1 binding to membranes: time-resolved studies of spin-labeled mutants.

To investigate the mechanism of interaction of the toxin colicin E1 with membranes, three cysteine substitution mutants and the wild type of the channel-forming fragment were spin labeled at the unique thiol. Time-resolved interaction of these labeled proteins with phospholipid vesicles was investigated with stopped-flow electron paramagnetic resonance spectroscopy. The fragment interacts with neutral bilayers at low pH, indicating that the interaction is hydrophobic rather than electrostatic. The interaction occurs in at least two distinct steps: (i) rapid adsorption to the surface; and (ii) slow, rate-limiting insertion of the hydrophobic central helices into the membrane interior.

Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants.

Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.

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.

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.

Docking phospholipase A2 on membranes using electrostatic potential-modulated spin relaxation magnetic resonance.

A method involving electron paramagnetic resonance spectroscopy of a site-selectively spin-labeled peripheral membrane protein in the presence and absence of membranes and of a water-soluble spin relaxant (chromium oxalate) has been developed to determine how bee venom phospholipase A2 sits on the membrane. Theory based on the Poisson-Boltzmann equation shows that the rate of spin relaxation of a protein-bound nitroxide by a membrane-impermeant spin relaxant depends on the distance (up to tens of angstroms) from the spin probe to the membrane. The measurements define the interfacial binding surface of this secreted phospholipase A2.

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.

Molecular characterization of helix-loop-helix peptides.

A class of regulators of eukaryotic gene expression contains a conserved amino acid sequence responsible for protein oligomerization and binding to DNA. This structure consists of an arginine- and lysine-rich basic region followed by a helix-loop-helix motif, which together mediate specific binding to DNA. Peptides were prepared that span this motif in the MyoD protein; in solution, they formed alpha-helical dimers and tetramers. They bound to DNA as dimers and their alpha-helical content increased on binding. Parallel and antiparallel four-helix models of the DNA-bound dimer were constructed. Peptides containing disulfide bonds were engineered to test the correctness of the two models. A disulfide that is compatible with the parallel model promotes specific interaction with DNA, whereas a disulfide compatible with the antiparallel model abolishes specific binding. Electron paramagnetic resonance (EPR) measurements of nitroxide-labeled peptides provided intersubunit distance measurements that also supported the parallel model.

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.

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.

Structure in the channel forming domain of colicin E1 bound to membranes: the 402-424 sequence.

To explore the structure of the pore-forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402-424. Each mutant was reacted with a sulfhydryl-specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406-416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402-420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408-R409, is located on the lipid-exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium.

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.

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.

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.

Site directed spin labeling studies of structure and dynamics in bacteriorhodopsin.

Site-directed spin labeling of membrane proteins has been used to determine: (1) the topography of the polypeptide chain with respect to the membrane/solution interface, and (2) the identity and orientation of secondary structure in selected regions. These features are deduced from the collision rates of nitroxide side chains with paramagnetic reagents in solution, and the principles of the method are reviewed with reference to bacteriorhodopsin. The dynamics of the nitroxide side chains relative to the backbone reveal tertiary interactions of the labeled site, and provide a promising means of time-resolving conformational changes. This aspect is illustrated by recent studies of structural changes in bacteriorhodopsin during the photocycle. In these experiments, nitroxide side chains were introduced at residues 72, 101 and 105 after replacement of the original residues by cysteine. Upon flash photolysis, the electron paramagnetic resonance spectrum of a nitroxide at 101, but not those at 72 or 105, is time-dependent. The spectral change develops during the decay of the M-intermediate, and reverses upon return to the ground state. The results suggest a movement of the C-D or E-F interhelical loops during the protonation changes of aspartate 96.

Spin labeled cysteines as sensors for protein-lipid interaction and conformation in rhodopsin.

In stoichiometric amounts, the spin label N-tempoyl-(p-chloromercuribenzamide) reacts rapidly with one cysteine residue in membrane-bound bovine rhodopsin. This residue is distinct from the two reactive cysteines previously used as attachment sites for spectroscopic labels, and is on the external surface of the protein near the cytoplasmic membrane/aqueous interface. The spin-labeled side chain has revealed a light-induced conformational change in membrane-bound rhodopsin that is apparently not associated with protein aggregation. The changes are reversible upon the addition of 11-cis retinal, and the magnitude of the change is dependent on the identity of the phospholipid in the surrounding bilayer. Alteration of lipid composition has a much larger effect on bleached rhodopsin than rhodopsin itself, indicating that the former is more readily deformable in response to changes in bilayer properties. This is consistent with the loss of 11-cis retinal binding energy in opsin compared to rhodopsin. These results provide direct structural evidence that the conformation of a membrane protein can be modulated by the lipid properties.

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.