NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively.Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.     

NMR solution structure of hPar14 reveals similarity to the peptidyl prolyl cis/trans isomerase domain of the mitotic regulator hPin1 but indicates a different functionality of the protein

The 131-amino acid residue parvulin-like human peptidyl-prolyl cis/trans isomerase (PPIase) hPar14 was shown to exhibit sequence similarity to the regulator enzyme for cell cycle transitions human hPin1, but specificity for catalyzing pSer(Thr)-Pro cis/trans isomerizations was lacking. To determine the solution structure of hPar14 the (1)H, (13)C, and (15)N chemical shifts of this protein have been assigned using heteronuclear two and three-dimensional NMR experiments on unlabeled and uniformly (15)N/(13)C-labeled recombinant protein isolated from Escherichia coli cells that overexpress the protein. The chemical shift assignments were used to interpret the NOE data, which resulted in a total of 1042 NOE restraints. The NOE restraints were used along with 71 dihedral angle restraints and 38 hydrogen bonding restraints to produce 50 low-energy structures. The hPar14 folds into a betaalpha(3)betaalphabeta(2) structure, and contains an unstructured 35-amino acid basic tail N-terminal to the catalytic core that replaces the WW domain of hPin1 homologs. The three-dimensional structures of hPar14 and the PPIase domain of human hPin1 reveal a high degree of conservation. The root-mean-square deviations of the mean atomic coordinates of the heavy atoms of the backbone between residues 38 to 45, 50 to 58, 64 to 70, 81 to 86, 115 to 119 and 122 to 128 of hPar14 were 0.81(+/-0.07) A. The hPar14 model structure provides insight into how this class of PPIases may select preferential secondary catalytic sites, and also allows identification of a putative DNA-binding motif in parvulin-like PPIases.     

NMR structure of the N-terminal domain of the replication initiator protein DnaA

DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 A based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.     

The three-dimensional structure of guanine-specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry.

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.     

Nuclear magnetic resonance solution structure of hirudin(1-51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain.

The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by 1H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1-51) was computed with the program DIANA and energy-minimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel beta-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II') and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 A for the backbone atoms and 0.77 A for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1-51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 A for the backbone atoms and 0.91 A for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1-51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.     

Structural comparison of monomeric variants of the chemokine MIP-1beta having differing ability to bind the receptor CCR5

MIP-1beta, a member of the chemokine family of proteins, tightly binds the receptor CCR5 as part of its natural function in the immune response, and in doing so also blocks the ability of many strains of HIV to enter the cell. The single most important MIP-1beta residue known to contribute to its interaction with the receptor is Phe13, which when mutated reduces the ability of MIP-1beta to bind to CCR5 by more than 1000-fold. To obtain a structural understanding of the dramatic effect of the absence of Phe13 in MIP-1beta, we used multidimensional heteronuclear NMR to determine the three-dimensional structure of the MIP-1beta F13A variant. We had previously shown that, unlike the wild-type protein which has been shown to be a tight dimer, the F13A mutant is monomeric even at high concentrations [Laurence, J. S., Blanpain, C., Burgner, J. W., Parmentier, M., and LiWang, P. J. (2000) Biochemistry 39, 3401-3409], leading to significant changes in the NMR spectra of F13A and the wild-type protein. We have obtained a total of 940 structural restraints for MIP-1beta F13A, and have calculated a family of structures having a backbone rmsd from the average of 0.55 A (residues 12-67). A structural comparison of the F13A mutant with a fully active monomeric variant, P8A, shows that despite some differences in the (1)H-(15)N HSQC spectra the two are nearly identical in NOE distance restraints and in backbone conformation. A comparison of F13A with the wild-type protein shows largely the same fold, although differences exist in the N-terminal and loop regions for which the loss of the dimer in F13A can mainly account. A dynamics comparison confirms greater flexibility in F13A than in the wild-type protein in regions of dimer contact in the wild-type protein. In an analysis to determine if the large functional effect resulting from the loss of Phe13 is due to the local side chain change or due to more global structural changes, we conclude that local effects predominate. This suggests that a strategy for designing tight binding anti-CCR5 therapeutics should include a Phe-like component.     

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein.

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.     

High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution

The solution structure of recombinant human thioredoxin (105 residues) has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Approximate interproton distance restraints were derived from nuclear Overhauser effect (NOE) measurements. In addition, a large number of stereospecific assignments for beta-methylene protons and torsion angle restraints for phi, psi, and chi 1 were obtained by using a conformational grid search on the basis of the intraresidue and sequential NOE data in conjunction with 3JHN alpha and 3J alpha beta coupling constants. The structure calculations were based on 1983 approximate interproton distance restraints, 52 hydrogen-bonding restraints for 26 hydrogen bonds, and 98 phi, 71 psi, and 72 chi 1 torsion angle restraints. The 33 final simulated annealing structures obtained had an average atomic rms distribution of the individual structures about the mean coordinate positions of 0.40 +/- 0.06 A for the backbone atoms and 0.78 +/- 0.05 A for all atoms. The solution structure of human thioredoxin consists of a five-stranded beta-sheet surrounded by four alpha-helices, with an active site protrusion containing the two redox-active cysteines. The overall structure is similar to the crystal and NMR structures of oxidized [Katti, S. K., LeMaster, D. M., & Eklund, H. (1990) J. Mol. Biol. 212, 167-184] and reduced [Dyson, J. H., Gippert, G. P., Case, D. A., Holmgren, A., & Wright, P. (1990) Biochemistry 29, 4129-4136] Escherichia coli thioredoxin, respectively, despite the moderate 25% amino acid sequence homology. Several differences, however, can be noted. The human alpha 1 helix is a full turn longer than the corresponding helix in E. coli thioredoxin and is characterized by a more regular helical geometry. The helix labeled alpha 3 in human thioredoxin has its counterpart in the 3(10) helix of the E. coli protein and is also longer in the human protein. In contrast to these structural differences, the conformation of the active site loop in both proteins is very similar, reflecting the perfect sequence identity for a stretch of eight amino acid residues around the redox-active cysteines.     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study

The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.     

The NMR side-chain assignments and solution structure of enzyme IIBcellobiose of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.     

NMR solution structure and receptor peptide binding of the CC chemokine eotaxin-2

The human CC chemokine eotaxin-2 is a specific agonist for the chemokine receptor CCR3 and may play a role in the recruitment of eosinophils in allergic diseases and parasitic infections. We report the solution structure of eotaxin-2 determined using heteronuclear and triple resonance NMR methods. A family of 20 structures was calculated by hybrid distance geometry-simulated annealing from 854 NOE distance restraints, 48 dihedral angle restraints, and 12 hydrogen bond restraints. The structure of eotaxin-2 (73 amino acid residues) consists of a helical turn (residues 17-20) followed by a 3-stranded antiparallel beta-sheet (residues 22-26, 37-41, and 44-49) and an alpha-helix (residues 54-66). The N-loop (residues 9-16) is packed against both the sheet and the helix with the two conserved disulfide bonds tethering the N-terminal/N-loop region to the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 7-66) are 0.52 and 1.13 A, respectively. A linear peptide corresponding to the N-terminal region of CCR3 binds to eotaxin-2, inducing concentration-dependent chemical shift changes or line broadening of many residues. The distribution of these residues suggests that the peptide binds into an extended groove located at the interface between the N-loop and the beta2-beta3 hairpin. The receptor peptide may also interact with the N-terminus of the chemokine and part of the alpha-helix. Comparison of the eotaxin-2 structure with those of related chemokines indicates several structural features that may contribute to receptor specificity.     

Sequence-specific 1H NMR assignments and determination of the secondary structure for the activation domain isolated from pancreatic procarboxypeptidase B

Nearly complete sequence-specific 1H NMR assignments are presented for amino acid residues 3-81 in the 81-residue globular activation domain of porcine pancreatic procarboxypeptidase B isolated after limited tryptic proteolysis of the zymogen. These resonance assignments are consistent with the chemically determined amino acid sequence. Regular secondary structure elements were identified from nuclear Overhauser effects and the sequence locations of slowly exchanging backbone amide protons. The molecule contains two alpha-helices, including residues 20-30 and approximately residues 58-72, and a three-stranded antiparallel beta-sheet with the individual strands extending approximately from 12 to 17, 50 to 55, and 75 to 77. The identification of these secondary structures and a preliminary analysis of additional long-range NOE distance constraints show that isolated activation domain B forms a stable structure with the typical traits of a globular protein. The data presented here are the basis for the determination of the complete three-dimensional structure of activation domain B, which is currently in progress.     

The influence of stereospecific assignments on the determination of three-dimensional structures of proteins by nuclear magnetic resonance spectroscopy. Application to the sea anemone protein BDS-I

The influence of the stereospecific assignments of beta-methylene protons and the classification of chi 1 torsion angles on the definition of the three-dimensional structures of proteins determined from NMR data is investigated using the sea anemone protein BDS-I (43 residues) as a model system. Two sets of structures are computed. The first set comprises 42 converged structures (denoted STEREO structures) calculated on the basis of the complete list of restraints derived from the NMR data, consisting of 489 interproton and 24 hydrogen bonding distance restraints, supplemented by 23 phi backbone and 21 chi 1 side chain torsion angle restraints. The second set comprises 31 converged structures (denoted NOSTEREO structures) calculated from a reduced data set in which those restraints arising from stereospecific assignments, and the corresponding chi 1 torsion angle restraints, are explicitly omitted. The results show that the inclusion of the stereospecific restraints leads to a significant improvement in the definition of the structure of BDS-I, both with respect to the backbone and the detailed arrangement of the side chains. Average atomic rms differences between the individual structures and the mean structures for the backbone atoms are 0.67 +/- 0.12 A and 0.93 +/- 0.16 A for the STEREO and NOSTEREO structures, respectively; the corresponding values for all atoms are 0.90 +/- 0.17 A and 1.17 +/- 0.17 A, respectively. In addition, while the overall fold remains unchanged, there is a small but significant atomic displacement between the two sets of structures.     

Structure and dynamics of micelle-associated human immunodeficiency virus gp41 fusion domain

The N-terminal fusion domain of the HIV-1 gp41 envelope glycoprotein is responsible for initiating the fusion of viral and cellular membranes, leading to the subsequent infection of the host cell by HIV-1. We have investigated the backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments using NMR spectroscopy and (15)N- and (15)N,(13)C,(2)H-labeled peptides. Similar (15)N-(1)H HSQC spectra were obtained in a variety of detergents, including SDS, DPC, mixed DPC/SDS, and LPPG micelles, indicating that the peptide structure is not strongly influenced by the type of detergent used. Detailed characterization was carried out in SDS micelles, where the long-term sample stability was found to be optimal. In addition to J-coupling and NOE restraints, a nearly complete set of backbone residual dipolar coupling restraints was recorded for the fusion domain-micelle complex aligned with respect to the magnetic field using a stretched polyacrylamide gel. Backbone amide (15)N spin relaxation and amide hydrogen exchange rates with the solvent were also measured. The ensemble of NMR structures reveals an uninterrupted alpha-helix for the least mobile residues (S(2) > 0.65), Ile-4 to Met-19, with transient helical character extending up to Ala-22. A 12-residue (Ile-4 to Ala-15) segment is fully shielded from solvent, with Gly-3 and Gly-16 found at micelle-solvent interfaces. Residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics relative to the residues buried in the micelle, and their mobility increases with the distance from the micelle.     

Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy

The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle restraints, and 17 hydrogen-bond distance restraints. The calculated structures contain two alpha-helices (residues 6-18 and 26-34) and a five-stranded antiparallel beta-sheet (residues 50-55, 70-75, 85-91, 94-101, and 105-108). The core of the protein is formed by the packing of one of the alpha-helices (residues 6-18) onto the beta-sheet. The average RMS deviation between the calculated structures and the mean structure is 1.11 A for the backbone atoms and 1.75 A for all atoms. The protein is least well-defined in the N-terminal region and in three large loops. When these regions are excluded, the average RMS deviation between the calculated structures and the mean structure for residues 5-34, 50-56, 71-76, 85-109 is 0.62 A for the backbone atoms and 1.0 A for all atoms. The NMR-derived structure has been compared with the crystal structure of barnase [Mauguen et al. (1982) Nature (London) 297, 162-164].