Structure and dynamics of Escherichia coli chemosensory receptors. Engineered sulfhydryl studies.

Cysteine residues introduced by site-directed mutagenesis have been used to probe the conformation and dynamics of two receptors in the E. coli chemotaxis pathway. (a) Thermal motions of the polypeptide backbone were investigated in the periplasmic D-galactose and D-glucose receptor, a globular protein of known structure. Disulfide bond formation between pairs of engineered sulfhydryls were used to trap collisions during the relative motions of surface alpha-helices I and X. Motions with amplitudes ranging from 4.5 to 15.2 A were detected on timescales ranging from 10(-4) to 10(-1) s, respectively. These results suggest that thermal backbone motions may have larger amplitudes than previously thought. (b) Conformational features of the transmembrane aspartate transducer have been investigated. Engineered sulfhydryls were used to ascertain the location and orientations of two putative transmembrane alpha-helices in the primary structure, to investigate the packing of these helices, to determine the oligomer and surface structures, and to detect thermal and ligand-induced dynamics of the polypeptide backbone. A model for the folded conformation of the transducer oligomer is reviewed.     

The aspartate receptor cytoplasmic domain: in situ chemical analysis of structure, mechanism and dynamics.

BACKGROUND: Site-directed sulfhydryl chemistry and spectroscopy can be used to probe protein structure, mechanism and dynamics in situ. The aspartate receptor of bacterial chemotaxis is representative of a large family of prokaryotic and eukaryotic receptors that regulate histidine kinases in two-component signaling pathways, and has become one of the best characterized transmembrane receptors. We report here the use of cysteine and disulfide scanning to probe the helix-packing architecture of the cytoplasmic domain of the aspartate receptor. RESULTS: A series of designed cysteine pairs have been used to detect proximities between cytoplasmic helices in the full-length, membrane-bound receptor by measurement of disulfide-bond formation rates. Upon mild oxidation, 25 disulfide bonds from rapidly between three specific pairs of helices, whereas other helix pairs yield no detectable disulfide-bond formation. Further constraints on helix packing are provided by 14 disulfide bonds that retain receptor function in an in vitro kinase regulation assay. Of these functional disulfides, seven lock the receptor in the conformation that constitutively stimulates kinase activity ('lock-on'), whereas the remaining seven retain normal kinase regulation. Finally, disulfide-trapping experiments in the absence of bound kinase reveal large-amplitude relative motions of adjacent helices, including helix translations and rotations of up to 19 A and 180 degrees, respectively. CONCLUSIONS: The 25 rapidly formed and 14 functional disulfide bonds identify helix-helix contacts and their register in the full-length, membrane-bound receptor-kinase complex. The results reveal an extended, rather than compact, domain architecture in which the observed helix-helix interactions are best described by a four-helix bundle arrangement. A cluster of six lock-on disulfide bonds pinpoints a region of the four-helix bundle critical for kinase activation, whereas the signal-retaining disulfides indicate that signal-induced rearrangements of this region are small enough to be accommodated by disulfide-bond flexibility (< or = 1.2 A). In the absence of bound kinase, helix packing within the cytoplasmic domain is highly dynamic.     

Signaling domain of the aspartate receptor is a helical hairpin with a localized kinase docking surface: cysteine and disulfide scanning studies.

Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related members of the taxis receptor family. Previous work has characterized the N-terminal section of the signaling domain [Bass, R. B., and Falke, J. J. (1998) J. Biol. Chem. 273, 25006-25014], while the present study focuses on the C-terminal section and the interactions between these two regions. Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit. The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic alpha-helix, denoted helix alpha8. The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419. Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation. Patterns of disulfide bond formation suggest that helix alpha8, together with the immediately N-terminal helix alpha7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices alpha7, alpha7', alpha8, and alpha8'. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices alpha7 and alpha8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking.     

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.     

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.     

Differential backbone dynamics of companion helices in the extended helical coiled‐coil domain of a bacterial chemoreceptor

Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four‐helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site‐directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent‐exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin‐labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane‐distal tight turn (N‐helix), and the other carboxyl terminal (C‐helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide‐spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N‐ and C‐helices. For bilayer‐inserted receptors, EPR spectra from sites in the membrane‐distal protein‐interaction region and throughout the C‐helix were typical of well‐structured helices. In contrast, for approximately two‐thirds of the N‐helix, from its origin as the AS‐2 helix of the membrane‐proximal HAMP domain to the beginning of the membrane‐distal protein‐interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four‐helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function.     

Evidence that the adaptation region of the aspartate receptor is a dynamic four-helix bundle: cysteine and disulfide scanning studies

The aspartate receptor is one of the ligand-specific, homodimeric chemoreceptors that detects extracellular attractants and triggers the chemotaxis pathway of Escherichia coli and Salmonella typhimurium. This receptor regulates the activity of the histidine kinase CheA, which forms a kinetically stable complex with the receptor cytoplasmic domain. An atomic four-helix bundle model has been constructed for this domain, which is functionally subdivided into the signaling and adaptation subdomains. The proposed four-helix bundle structure of the signaling subdomain, which binds CheA, is fully supported by experimental evidence. Much less evidence is available to test the four-helix bundle model of the adaptation subdomain, which possesses covalent adaptation sites and docking surfaces for adaptation enzymes. The present study focuses on a putative helix near the C terminus of the adaptation subdomain. To probe the structural and functional features of positions G467-A494 in this C-terminal region, a cysteine and disulfide scanning approach has been employed. Measurement of the chemical reactivities of scanned cysteines reveals an alpha-helical periodicity of exposed and buried residues, confirming alpha-helical secondary structure and mapping out a buried packing face. The effects of cysteine substitutions on activity in vivo and in vitro highlight the functional importance of the helix, especially its buried face. A scan for disulfide bond formation between symmetric pairs of engineered cysteines reveals promiscuous collisions between subunits, indicating the presence of significant thermal dynamics. A scan for functional disulfides reveals lock-on and signal-retaining disulfide bonds formed between symmetric pairs of cysteines at buried positions, indicating that the buried face of the helix lies near the subunit interface of the homodimer in the equilibrium structures of both the apo and aspartate-bound states where it plays a critical role in kinase regulation. These results strongly support the existing four-helix bundle model of the adaptation subdomain structure. A mechanistic model is proposed in which a signal is transmitted through the adaptation subdomain by a change in supercoiling of the four-helix bundle.     

Structure and dynamics of an amphiphilic peptide in a lipid bilayer: a molecular dynamics study

A molecular dynamics simulation of a simple model membrane system composed of a single amphiphilic helical peptide (ace-K2GL16K2A-amide) in a fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer was performed for a total of 1060 ps. The secondary structure of the peptide and its stability were described in terms of average dihedral angles, phi and psi, and the C alpha torsion angles formed by backbone atoms; by the average translation per residue along the helix axis; and by the intramolecular peptide hydrogen bonds. The results indicated that residues 6 through 15 remain in a stable right-handed alpha-helical conformation, whereas both termini exhibit substantial fluctuations. A change in the backbone dihedral angles for residues 16 and 17 is accompanied by the loss of two intramolecular hydrogen bonds, leading to a local but long-lived disruption of the helix. The dynamics of the peptide was characterized in terms of local and global helix motions. The local motions of the N-H bond angles were described in terms of the autocorrelation functions of P2[cos thetaNH(t, t + tau)] and reflected the different degrees of local peptide order as well as a variation in time scale for local motions. The chi1 and chi2 dihedral angles of the leucine side chains underwent frequent transitions between potential minima. No connection between the side-chain positions and their mobility was observed, however. In contrast, the lysine side chains displayed little mobility during the simulation. The global peptide motions were characterized by the tilting and bending motions of the helix. Although the peptide was initially aligned parallel to the bilayer normal, during the simulation it was observed to tilt away from the normal, reaching an angle of approximately 25 degrees by the end of the simulation. In addition, a slight bend of the helix was detected. Finally, the solvation of the peptide backbone and side-chain atoms was also investigated.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Molecular motions within the pore of voltage-dependent sodium channels.

The pores of ion channel proteins are often modeled as static structures. In this view, selectivity reflects rigidly constrained backbone orientations. Such a picture is at variance with the generalization that biological proteins are flexible, capable of major internal motions on biologically relevant time scales. We tested for motions in the sodium channel pore by systematically introducing pairs of cysteine residues throughout the pore-lining segments. Two distinct pairs of residues spontaneously formed disulfide bonds bridging domains I and II. Nine other permutations, involving all four domains, were capable of disulfide bonding in the presence of a redox catalyst. The results are inconsistent with a single fixed backbone structure for the pore; instead, the segments that line the permeation pathway appear capable of sizable motions.     

Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component

The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane alpha-helix that precedes an amphipathic alpha-helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.     

Determination of the molecular dynamics of alamethicin using 13C NMR: implications for the mechanism of gating of a voltage-dependent channel.

Alamethicin is a channel-forming peptide antibiotic that produces a highly voltage-dependent conductance in planar bilayers. To provide insight into the mechanisms for its voltage dependence, the dynamics of the peptide were examined in solution using nuclear magnetic resonance. Natural-abundance 13C spin-lattice relaxation rates and 13C-1H nuclear Overhauser effects of alamethicin were measured at two magnetic field strengths in methanol. This information was interpreted using a model-free approach to obtain values for the overall correlation times as well as the rates and amplitudes of the internal motions of the peptide. The picosecond, internal motions of alamethicin are highly restricted along the peptide backbone and indicate that it behaves as a rigid helical rod in solution. The side chain carbons exhibit increased segmental motion as their distance from the peptide backbone is increased; however, these motions are not unrestricted. Methyl group dynamics are also consistent with the restricted motions observed for the backbone carbons. There is no evidence from these dynamics measurements for a hinged motion of the peptide about proline-14. Alamethicin appears to be slightly less structured in methanol than in the membrane; as a result, alamethicin is also expected to behave as a rigid helix in the membrane. This suggests that the gating of this peptide involves changes in the orientation of the entire helix, rather than the movement of a segment of the peptide backbone.     

The proximity between helix I and helix XI in the melibiose carrier of Escherichia coli as determined by cross-linking

The melibiose carrier of Escherichia coli is a transmembrane protein that comprises 12 transmembrane helices connected by periplasmic and cytoplasmic loops, with both the N- and C-termini located on the cytoplasmic side. Our previous studies of second-site revertants suggested proximity between several helices, including helices XI and I. In this study, we constructed six double cysteine mutants, each having one cysteine in helix I and the other in helix XI: three mutants, K18C/S380C, D19C/S380C, and F20C/S380C, have their cysteine pairs near the cytoplasmic side of the carrier, and the other three, T34C/G395C, D35C/G395C, and V36C/G395C, have their cysteine pairs near the periplasmic side. In the absence of substrate, disulfide formations catalyzed by iodine and copper-(1,10-phenanthroline)(3) indicate that helix I and helix XI are in immediate proximity to each other on the periplasmic side but not on the cytoplasmic side, as shown by protease cleavage analyses. We infer that the two helices are tilted with respect to each other, with the periplasmic sides in close proximity.     

Relative proximity and orientation of helices 4 and 8 of the GLUT1 glucose transporter

A structure has been proposed for glucose transporter-1 (GLUT1) based upon homology modeling that is consistent with the results of numerous mutagenesis studies (Mueckler, M., and Makepeace, C. (2004) J. Biol. Chem. 279, 10494-10499). To further test and refine this model, the relative orientation and proximity of transmembrane helices 4 and 8 were analyzed by chemical crosslinking of di-cysteine mutants created in a reporter GLUT1 construct. All six native cysteine residues of GLUT1 were changed to either glycine or serine residues by site-directed mutagenesis, resulting in a functional Glut1 construct with Cys mutated to Gly/Ser (C-less). The GLUT1 reporter molecule was engineered from C-less GLUT1 by creating a unique cleavage site for factor Xa protease within the central cytoplasmic loop and by eliminating the site of N-linked glycosylation. Fourteen functional di-cysteine mutants were then created from the C-less reporter construct, each mutant containing a single cysteine residue in helix 4 and one cysteine residue in helix 8. These mutants were expressed in Xenopus oocytes, and the sensitivity of each mutant to intramolecular crosslinking by two homo-bifunctional, thiol-specific crosslinking reagents, bismaleimidehexane and 1,4-phenylenedimaleimide, was ascertained by protease cleavage followed by immunoblot analysis. Four pairs of cysteine residues, Cys(148)/Cys(328), Cys(145)/Cys(328), Cys(148)/Cys(325), and Cys(145)/Cys(325), were observed to be in close enough proximity to be susceptible to crosslinking by one or both reagents. All five of the cysteine residues susceptible to crosslinking are predicted to lie on the same face of helix 4 or 8 and to reside close to the cytoplasmic face of the membrane. These data indicate that the cytoplasmic ends of helices 4 and 8 lie within 6-16 A of one another and that the two helices twist or tilt such that they are further than 16 A apart toward the center and the exoplasmic side of the membrane. An updated model for the clustering of the transmembrane helices of GLUT1 is presented based on these data.     

Four free cysteine residues found in human IgG1 of healthy donors

Modifications with different thiol reagents demonstrated that 28 of 32 cysteine residues of human IgG1 are involved in the formation of disulfide bonds, and four cysteines remain free. So IgG1 is a protein possessing both free SH-groups and disulfide bonds. Only one of the four SH-groups is accessible for silver or mercury ions and hydrophobic reagents, whereas the remaining three SH-groups are masked and can be revealed only after deep denaturation of the protein. Detection of the masked cysteine residues was shown to depend on the kinetics of intramolecular changes occurring during denaturation of the protein and on the method of the assay of the SH-groups.     

Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation

Oxidation of cysteine pairs to disulfide requires cellular factors present in the bacterial periplasmic space. DsbB is an E. coli membrane protein that oxidizes DsbA, a periplasmic dithiol oxidase. To gain insight into disulfide bond formation, we determined the crystal structure of the DsbB-DsbA complex at 3.7 A resolution. The structure of DsbB revealed four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. We propose that DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.     

Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.     

NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.