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

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

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

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

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

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

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

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

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

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

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

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

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

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

Identifying conformational changes with site-directed spin labeling.

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

Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling.

Side chain mobility, accessibility, and backbone motion were studied by site-directed spin labeling of sequential cysteine mutants of the G strand in tear lipocalins (TL). A nitroxide scan between residues 98 and 105 revealed the alternating periodicity of mobility and accessibility to NiEDDA and oxygen, characteristic of a beta-strand. Residue 99 was the most inaccessible to NiEDDA and oxygen. EPR spectra with the fast relaxing agent, K(3)Fe(CN)(6), exhibited two nitroxide populations for most residues. The motionally constrained population was relatively less accessible to K(3)Fe(CN)(6) because of dynamic tertiary contact, probably with side chain residues of adjacent strands. With increasing concentrations of sucrose, the spectral contribution of the immobile component was greater, indicating a larger population with tertiary contact. Increased concentrations of sucrose also resulted in a restriction of mobility of spin-labeled fatty acids which were bound within the TL cavity. The data suggest that sucrose enhanced ligand affinity by slowing the backbone motion of the lipocalin. The correlation time of an MTSL derivative (I) attached to F99C resulted in the lack of side chain motion and therefore reflects the overall rotation of the TL complex. The correlation time of F99C in tears (13.5 ns) was the same as that in buffer and indicates that TL exists as a dimer under native conditions. TL-spin-labeled ligand complexes have a shorter correlation time than the protein alone, indicating that the fatty acids are not rigidly anchored in the cavity, but move within the pocket. This segmental motion of the ligand was modulated by protein backbone fluctuations. Accessibility studies with oxygen and NiEDDA were performed to determine the orientation and depth of a series of fatty acid derivatives in the cavity of TL. Fatty acids are oriented with the hydrocarbon tail buried in the cavity and the carboxyl group oriented toward the mouth. In general, the mobility of the nitroxide varied according to position such that nitroxides near the mouth had greater mobility than those located deep in the cavity. Nitroxides positioned up to 16 carbon units from the hydrocarbon tail of the ligand are motionally restricted and inaccessible, indicating the cavity extends to at least this depth. EPR spectra obtained with and without sucrose showed that the intracavitary position of lauric acid in TL is similar to that in beta-lactoglobulin. However, unlike beta-lactoglobulin, TL binds 16-doxyl stearic acid, suggesting less steric hindrance and greater promiscuity for TL.

Structure of the KcsA potassium channel from Streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment.

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.

Conformation of the diphtheria toxin T domain in membranes: a site-directed spin-labeling study of the TH8 helix and TL5 loop.

The isolated T domain of diphtheria toxin was mutated by cysteine-scanning mutagenesis at 28 consecutive sites (residues 328-355) that comprise the TH8 helix and the TL5 interhelical loop in the native toxin. After derivatizing the mutant proteins with a sulfhydryl-selective nitroxide reagent, we examined the mobility of each nitroxide and its accessibility to polar and nonpolar paramagnetic reagents, before and after insertion into phospholipid bilayers. The data obtained with the proteins in solution at pH 8 are generally consistent with predictions from the crystal structure of the toxin. Upon membrane binding at pH 4.6, a major structural reorganization of the domain was seen, which dramatically reduced the accessibility of most residues in this region to the polar reagent nickel(II)-ethylenediaminediacetate complex (NiEDDA). Many of these residues also showed reduced accessibility to the nonpolar reagent O(2). Periodic accessibility of the nitroxide side chains along the sequence to these reagents shows that TH8 remains largely helical in the membrane-bound state, with one surface associated with protein and the other facing the hydrophobic interior of the bilayer. In addition, the TL5 loop also appears to become alpha-helical in the membrane, with one surface in contact with protein and the other in contact with the bilayer interior. These findings provide a structural framework for understanding how the T domain forms a transmembrane channel and mediates translocation of diphtheria toxin's enzymic moiety across a membrane.

Structural features of the C-terminal domain of bovine rhodopsin: a site-directed spin-labeling study.

Eleven single-cysteine substitution mutants have been prepared in the sequence 325-340 of rhodopsin, corresponding to the C-terminal domain. Each of the cysteine mutants was modified with a selective nitroxide reagent to introduce a spin-labeled side chain. The electron paramagnetic resonance spectra of the labeled proteins were analyzed in terms of side chain dynamics. At all sites, the spectra reflected the presence of two populations of different mobility, although one was always dominant. The mobility of the dominant population increased in a regular fashion from the palmitoylation sites at 322C and 323C to the C-terminus, where the spectra resembled those of an unfolded protein. This apparent mobility gradient is only slightly affected in mutants lacking the palmitoyl groups, suggesting that they are not responsible for physically anchoring the C-terminal peptide at one end. Binding of a monoclonal antibody to its epitope at the C-terminus dramatically reduces the mobility of nearby residues, creating a local mobility gradient opposite that in the absence of the antibody. These results indicate that the C-terminal domain of rhodopsin, beyond the palmitoylation sites, is highly disordered and dynamic, resembling an unfolded peptide tethered at one end.

Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study.

Sixteen single-cysteine substitution mutants of rhodopsin were prepared in the sequence 306-321 which begins in transmembrane helix VII and ends at the palmitoylation sites at 322C and 323C. The substituted cysteine residues were modified with a selective reagent to generate a nitroxide side chain, and the electron paramagnetic resonance spectrum of each spin-labeled mutant was analyzed in terms of residue accessibility and mobility. The periodic behavior of these parameters along the sequence indicated that residues 306-314 were in a regular alpha-helical conformation representing the end of helix VII. This helix apparently extends about 1.5 turns above the surface of the membrane, with one face in strong tertiary interaction with the core of the protein. For the segment 315-321, substituted cysteine residues at 317, 318, 320, and 321 had low reactivity with the spin-label reagent. This segment has the most extensive tertiary interactions yet observed in the rhodopsin extra-membrane sequences at the cytoplasmic surface. Previous studies showed the spontaneous formation of a disulfide bond between cysteine residues at 65 and 316. This result indicates that at least some of the tertiary contacts made in the 315-321 segment are with the sequence connecting transmembrane helices I and II. Photoactivation of rhodopsin produces changes in structure detected by spin labels at 306, 313, and 316. The changes at 313 can be accounted for by movements in the adjacent helix VI.

Single-cysteine substitution mutants at amino acid positions 306-321 in rhodopsin, the sequence between the cytoplasmic end of helix VII and the palmitoylation sites: sulfhydryl reactivity and transducin activation reveal a tertiary structure.

As sensors for structure at the cytoplasmic face of rhodopsin, single-cysteine substitution mutants have been previously studied in the regions connecting helices III and IV and helices V and VI. In this paper we report on single-cysteine substitution mutants at amino acid positions 306-321, comprising the cytoplasmic sequence between helix VII and the palmitoylation sites in rhodopsin. The cysteine opsin mutants were expressed in COS-1 cells and on treatment with 11-cis-retinal all formed the characteristic rhodopsin chromophore. Cysteines at positions 306-316 and 319 reacted in the dark with the thiol-specific reagent 4, 4'-dithiodipyridine (4-PDS) but showed a wide variation in reactivity. Cysteines at positions 317, 318, 320, and 321 showed no reaction with 4-PDS. The mutants on illumination also showed wide variations in activating GT. The mutant Y306C showed almost no GT activation, I307C and N310C were poor, and the activity of the mutants M309C, F313C, and M317C was also reduced relative to WT. The results suggest that the region comprising amino acids 306-321 is a part of a tertiary structure and that specific amino acids in this region on light-activation participate in the interaction with GT.

Single-cysteine substitution mutants at amino acid positions 55-75, the sequence connecting the cytoplasmic ends of helices I and II in rhodopsin: reactivity of the sulfhydryl groups and their derivatives identifies a tertiary structure that changes upon light-activation.

Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.

Structural features and light-dependent changes in the sequence 59-75 connecting helices I and II in rhodopsin: a site-directed spin-labeling study.

Twenty-one single-cysteine substitution mutants were prepared in the sequence 56-75 between transmembrane helices I and II at the cytoplasmic surface of bovine rhodopsin. Each mutant was reacted with a sulfhydryl-specific reagent to produce a nitroxide side chain. The electron paramagnetic resonance of the labeled proteins in dodecyl maltoside solution was analyzed to provide the relative mobility and accessibility of the nitroxide side chain to both polar and nonpolar paramagnetic reagents. The results indicate that the hydrophobic-water interface of the micelle intersects helices I and II near residues 64 and 71, respectively. Thus, the sequence 64-71 is in the aqueous phase, while 56-63 and 72-75 lie in the transmembrane helices I and II, respectively. The lipid-facing surfaces on transmembrane helices I and II near the cytoplasmic surface correspond to approximately 180 degrees and 90 degrees of arc on the helical surfaces, respectively. Photoactivation of rhodopsin produced changes in structure in the region investigated, primarily around helix II. However, these changes are much smaller than those noted by spin labels in helix VI (Altenbach, C., Yang, K., Farrens, D., Farahbakhsh, Z., Khorana, H. G., and Hubbell, W. L. (1996) Biochemistry 35, 12470).

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

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

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.

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.