Structural basis for the photochemistry of alpha-phycoerythrocyanin

Phycobiliproteins and phytochromes are light-harvesting and light-sensing proteins containing linear tetrapyrroles, so-called bile chromophores. The chromophores in certain biliproteins, including the phytochromes, isomerize reversibly from a stable Z-configuration to a stable E-configuration when irradiated with light of the appropriate wavelength. Here, we report the crystal structure of alpha-phycoerythrocyanin with its chromophore in the E-configuration, compare it with the Z-configuration found in trimeric phycoerythrocyanin, and reveal the structural bases of the isomerization. The geometric changes of the chromophore account for the large spectral shift, which characterizes the overall transition. Interactions of the chromophore A and D pyrrole rings with flexible protein moieties are required for the formation and stabilization of the isomers. We predict that the results will hold for all photoactive biliproteins.     

Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.     

Intrinsic Disorder of the Neuronal SNARE Protein SNAP25a in its Pre-fusion Conformation

The neuronal SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze monomeric SNAP25a by NMR spectroscopy, further supported by small-angle X-ray scattering (SAXS) experiments. SAXS data indicate that monomeric SNAP25 is more compact than a Gaussian chain but still a random coil. NMR shows that for monomeric SNAP25a, before SNAP25a interacts with its SNARE partners to drive membrane fusion, only the N-terminal part (region A5 to V36) of the first SNARE motif, SN1 (L11 - L81), is helical, comprising two α-helices (ranging from A5 to Q20 and S25 toV36). From E37 onwards, SNAP25a is mostly disordered and displays high internal flexibility, including the C-terminal part of SN1, almost the entire second SNARE motif (SN2, N144-A199), and the connecting loop region. Apart from the N-terminal helices, only the C-termini of both SN1 (E73 - K79) and SN2 (region T190 - A199), as well as two short regions in the connecting loop (D99 - K102 and E123 - M127) show a weak α-helical propensity (α-helical population < 25%). We speculate that the N-terminal helices (A5 to Q20 and S25 to V36) which constitute the N-terminus of SN1 act as a nucleation site for initiating SNARE zippering.     

Three-dimensional solution structure of Ca(2+)-loaded porcine calbindin D9k determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of native, intact porcine calbindin D9k has been determined by distance geometry and restrained molecular dynamics calculations using distance and dihedral angle constraints obtained from 1H NMR spectroscopy. The protein has a well-defined global fold consisting of four helices oriented in a pairwise antiparallel manner such that two pairs of helix-loop-helix motifs (EF-hands) are joined by a linker segment. The two EF-hands are further coupled through a short beta-type interaction between the two Ca(2+)-binding loops. Overall, the structure is very similar to that of the highly homologous native, minor A form of bovine calbindin D9k determined by X-ray crystallography [Szebenyi, D. M. E., & Moffat, K. (1986) J. Biol. Chem. 261, 8761-8776]. A model structure built from the bovine calbindin D9k crystal structure shows several deviations larger than 2 A from the experimental distance constraints for the porcine protein. These structural differences are efficiently removed by subjecting the model structure to the experimental distance and dihedral angle constraints in a restrained molecular dynamics protocol, thereby generating a model that is very similar to the refined distance geometry derived structures. The N-terminal residues of the intact protein that are absent in the minor A form appear to be highly flexible and do not influence the structure of other regions of the protein. This result is important because it validates the conclusions drawn from the wide range of studies that have been carried out on minor A forms rather than the intact calbindin D9k.     

Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Time-resolved resonance raman structural studies of the pB' intermediate in the photocycle of photoactive yellow protein

Time-resolved resonance Raman spectroscopy is used to obtain chromophore vibrational spectra of the pR, pB', and pB intermediates during the photocycle of photoactive yellow protein. In the pR spectrum, the C8-C9 stretching mode at 998 cm(-1) is approximately 60 cm(-1) lower than in the dark state, and the combination of C-O stretching and C7H=C8H bending at 1283 cm(-1) is insensitive to D2O substitution. These results indicate that pR has a deprotonated, cis chromophore structure and that the hydrogen bonding to the chromophore phenolate oxygen is preserved and strengthened in the early photoproduct. However, the intense C7H=C8H hydrogen out-of-plane (HOOP) mode at 979 cm(-1) suggests that the chromophore in pR is distorted at the vinyl and adjacent C8-C9 bonds. The formation of pB' involves chromophore protonation based on the protonation state marker at 1174 cm(-1) and on the sensitivity of the COH bending at 1148 cm(-1) as well as the combined C-OH stretching and C7H=C8H bending mode at 1252 cm(-1) to D2O substitution. The hydrogen out-of-plane Raman intensity at 985 cm(-1) significantly decreases in pB', suggesting that the pR-to-pB' transition is the stage where the stored photon energy is transferred from the distorted chromophore to the protein, producing a more relaxed pB' chromophore structure. The C=O stretching mode downshifts from 1660 to 1651 cm(-1) in the pB'-to-pB transition, indicating the reformation of a hydrogen bond to the carbonyl oxygen. Based on reported x-ray data, this suggests that the chromophore ring flips during the transition from pB' to pB. These results confirm the existence and importance of the pB' intermediate in photoactive yellow protein receptor activation.     

Solution structure of the eukaryotic pore-forming cytolysin equinatoxin II: implications for pore formation

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. The structure of the actinoporin equinatoxin II in aqueous solution, determined from NMR data, consists of two short helices packed against opposite faces of a beta-sandwich structure formed by two five-stranded beta-sheets. The protein core has extensive hydrophobic interfaces formed by residues projecting from the internal faces of the two beta-sheets. 15N relaxation data show uniform backbone dynamics, implying that equinatoxin II in solution is relatively rigid, except at the N terminus; its inferred rotational correlation time is consistent with values for monomeric proteins of similar mass. Backbone amide exchange rate data also support the view of a stable structure, even though equinatoxin II lacks disulfide bonds. As monitored by NMR, it unfolds at around 70 degrees C at pH 5.5. At 25 degrees C the structure is stable over the pH range 2.5-7.3 but below pH 2.5 it undergoes a slow transition to an incompletely unfolded structure resembling a molten globule. Equinatoxin II has two significant patches of positive electrostatic potential formed by surface-exposed Lys and Arg residues, which may assist its interaction with charged regions of the lipid head groups. Tyr and Trp residues on the surface may also contribute by interacting with the carbonyl groups of the acyl chains of target membranes. Data from mutational studies and truncated analogues identify two regions of the protein involved in membrane interactions, the N-terminal helix and the Trp-rich region. Once the protein is anchored, the N-terminal helix may penetrate the membrane, with up to four helices lining the pore, although other mechanisms of pore formation cannot be ruled out.     

Folding determinants of disulfide bond forming protein B explored by solution nuclear magnetic resonance spectroscopy

The two-stage model for membrane protein folding postulates that individual helices form first and are subsequently packed against each other. To probe the two-stage model, the structures of peptides representing individual transmembrane helices of the disulfide bond forming protein B have been studied in trifluoroethanol solution as well as in detergent micelles using nuclear magnetic resonance (NMR) and circular dichroism spectroscopy. In TFE solution, peptides showed well-defined α-helical structures. Peptide structures in TFE were compared to the structures of full-length protein obtained by X-ray crystallography and NMR. The extent of α-helical secondary structure coincided well, lending support for the two-stage model for membrane protein folding. However, the conformation of some amino acid side chains differs between the structures of peptide and full-length protein. In micellar solution, the peptides also adopted a helical structure, albeit of reduced definition. Using measurements of paramagnetic relaxation enhancement, peptides were confirmed to be embedded in micelles. These observations may indicate that in the native protein, tertiary interactions additionally stabilize the secondary structure of the individual transmembrane helices.     

The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.     

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.     

A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands

BACKGROUND: THP12 is an abundant and extraordinarily hydrophilic hemolymph protein from the mealworm Tenebrio molitor and belongs to a group of small insect proteins with four highly conserved cysteine residues. Despite their sequence homology to odorant-binding proteins and pheromone-binding proteins, the function of these proteins is unclear. RESULTS: The first three-dimensional structure of THP12 has been determined by multidimensional NMR spectroscopy. The protein has a nonbundle helical structure consisting of six alpha helices. The arrangement of the alpha helices has a 'baseball glove' shape. In addition to the hydrophobic core, electrostatic interactions make contributions to the overall stability of the protein. NMR binding studies demonstrated the binding of small hydrophobic ligands to the single hydrophobic groove in THP12. Comparing the structure of THP12 with the predicted secondary structure of homologs reveals a common fold for this new class of insect proteins. A search with the program DALI revealed extensive similarity between the three-dimensional structure of THP12 and the N-terminal domain (residues 1-95) of recoverin, a member of the family of calcium-binding EF-hand proteins. CONCLUSIONS: Although the biological function of this new class of proteins is as yet undetermined, a general role as alpha-helical carrier proteins for small hydrophobic ligands, such as fatty acids or pheromones, is proposed on the basis of NMR-shift perturbation spectroscopy.     

1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-gamma.

1H, 13C, and 15N NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz 13C alpha and 1H alpha line widths of this high molecular weight dimer and the extensive overlap in the 1H alpha and 13C alpha spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the 15N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the 15N-separated 3D NOESY-HMQC and 13C/15N-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C alpha and C beta chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-702], i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.     

Probing the nature of the blue-shifted intermediate of photoactive yellow protein in solution by NMR: hydrogen-deuterium exchange data and pH studies

The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded. Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data. It is likely that in the crystalline environment the ordered form of pB is strongly favored.     

An amphipathic helical region of the N-terminal barrel of phospholipid transfer protein is critical for ABCA1-dependent cholesterol efflux

Phospholipid lipid transfer protein (PLTP) mimics high-density lipoprotein apolipoproteins in removing cholesterol and phospholipids from cells through the ATP-binding cassette transporter A1 (ABCA1). Because amphipathic alpha-helices are the structural determinants for ABCA1 interactions, we examined the ability of synthetic peptides corresponding to helices in PLTP to remove cellular cholesterol by the ABCA1 pathway. Of the seven helices tested, only one containing PLTP residues 144-163 (p144), located at the tip of the N-terminal barrel, promoted ABCA1-dependent cholesterol efflux and stabilized ABCA1 protein. Mutating methionine 159 (Met-159) in this helix in PLTP to aspartate (M159D) or glutamate (M159E) nearly abolished the ability of PLTP to remove cellular cholesterol and dramatically reduced PLTP binding to phospholipid vesicles and its phospholipid transfer activity. These mutations impaired PLTP binding to ABCA1-generated lipid domains and PLTP-mediated stabilization of ABCA1 but increased PLTP binding to ABCA1. PLTP interactions with ABCA1 also mimicked apolipoproteins in activating Janus kinase 2; however, the M159D/E mutants were also able to activate this kinase. Structural analyses showed that the M159D/E mutations had only minor effects on PLTP conformation. These findings indicate that PLTP helix 144-163 is critical for removing lipid domains formed by ABCA1, stabilizing ABCA1 protein, interacting with phospholipids, and promoting phospholipid transfer. Direct interactions with ABCA1 and activation of signaling pathways likely involve other structural determinants of PLTP.     

Spectral Tuning of the Photoactive Yellow Protein Chromophore by H-Bonding

Spectral tuning in the photoactive yellow protein (PYP) is investigated by performing gas-phase absorption measurements on a PYP-model chromophore with two water molecules hydrogen-bonded to it. The photoabsorption maximum shows an unusually large blue shift of 0.71 eV in going from the bare to the hydrogen-bonded chromophore. It is concluded that several interactions within the PYP protein are mutually canceling each other, yielding an absorption maximum that is close to the absorption maximum of the bare chromophore. The system breaks apart upon photoexcitation in the gas phase by releasing the two water molecules, leaving the chromophore itself intact. The hydrogen-bonding interactions thus play an important role in stabilizing the gas phase chromophore against photofragmentation. The relaxation dynamics for the breakup process was also studied, and the timescale of relaxation via fragmentation was found to be <25 ns.     

Solid-state NMR spectroscopic study of chromophore-protein interactions in the Pr ground state of plant phytochrome A

Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A (phyA), the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional (2-D) magic-angle spinning solid-state NMR experiments on the recombinant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat (Avena sativa), assembled with uniformly 13C- and 15N-labeled phycocyanobilin (u-[13C,15N]-PCB-As.phyA3). The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D 13C-13C and 1H-15N experiments, a complete set of 13C and 15N assignments for the chromophore were obtained. Also, a large number of 1H-13C distance restraints between the chromophore and its binding pocket were revealed by interfacial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carboxylate moiety, together with the observation of two Pr isoforms, Pr-I and Pr-II, demonstrate the local mobility of the chromophore and the plasticity of its protein environment. It appears that the interactions and dynamics in the binding pocket of phyA in the Pr state are remarkably similar to those of cyanobacterial phytochrome (Cph1). The N-terminus of the region modeled (residues 56-66 of phyA) is highly mobile. Differences in the regulatory processes involved in plant and Cph1 phytochromes are discussed.     

Nuclear magnetic resonance solution structure of truncated human GRObeta [5-73] and its structural comparison with CXC chemokine family members GROalpha and IL-8

The three-dimensional structure of a novel four amino acid truncated form of the CXC chemokine GRObeta [5-73] isolated from bone marrow stromal cells with potent hematopoietic and anti-infective activities has been determined by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 1878 upper distance constraints derived from nuclear Overhauser effects (NOE) and 314 dihedral angle constraints, a group of 20 conformers representing the solution structure of the human GRObeta [5-73] was computed with the program DYANA. At the concentrations used for NMR study, GRObeta [5-73] forms a dimer in solution that is architectured by a six-stranded antiparallel beta-sheet (residues 25 to 29, 39 to 44, 49 to 52) and a pair of helices (residues 58 to 68) with 2-fold symmetry, while the C terminus of the protein is disordered. The average of the pairwise root-mean-square deviations of individual NMR conformers relative to the mean coordinates for the backbone atoms N, C(alpha) and C' of residues 5 to 68 is 0.47 A. Overall, the global fold of GRObeta [5-73] is similar to that of the previously reported NMR structure of GROalpha and the NMR and X-ray structures of interleukin-8. Among these three CXC chemokines, GRObeta [5-73] is most similar in structure to GROalpha. Significant differences between GRObeta [5-73], GROalpha and interleukin-8 are in the N-terminal loop comprising residues 12 to 19. The N-terminal arm containing the conserved ELR motif and the loop of residues 30 to 38 containing the GPH motif are different among these three CXC chemokines. The structural differences in these two regions may be responsible for the specificity of the receptor binding and biological activity of different chemokines.     

A topological model of the interaction between alpha-synuclein and sodium dodecyl sulfate micelles

Human alpha-synuclein is a 140-amino acid protein of unknown function abundantly expressed in the brain and found in Lewy bodies, a characteristic feature of Parkinson's disease. Alpha-synuclein is random in water under physiological conditions, but the first approximately 100 residues interact with SDS micelles or acidic phospholipid small unilamellar vesicles and adopt an ordered conformation. The rest of the molecule remains disordered in the bulk of the solution. The conformation of the N-terminal portion of the molecule in lipids was described as an extended helix [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926], as two distinct alpha-helices interrupted by a two-residue break [Chandra, S., Chen, X., Rizo, J., Jahn, R., and Sudhof, T. C. (2003) J. Biol. Chem. 278, 15313-15318], or as a noncanonical conformation, the alpha11/3 helix [Bussell, R., Jr., and Eliezer, D. (2003) J. Mol. Biol. 329, 763-778]. We characterized the topology of the different regions of alpha-synuclein relative to the surface of SDS micelles using spin probe-induced broadening of NMR signals, (15)N relaxation measurements, and fluorescence spectroscopy. Our results support the presence of two N-terminal helices, positioned on the surface of the micelle and separated by a flexible stretch. The region of residues 61-95 of the protein also adopts a helical conformation, but it is partially embedded in the micelle. These results could shed some light on the role of the membrane on the aggregation process of alpha-synuclein.     

Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.