The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.     

Determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing

The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.     

Solution conformation of thymosin beta 4: a nuclear magnetic resonance and simulated annealing study

The conformation of the polypeptide thymosin beta 4 in solutions of 60% (v/v) trifluoroethanol-d3 and 50% (v/v) hexafluoroisopropyl-d2 alcohol in water is investigated by nuclear magnetic resonance (NMR) spectroscopy. Under these conditions thymosin beta 4 adopts an ordered structure. By use of a combination of two-dimensional NMR techniques, the 1H NMR spectrum of thymosin beta 4 is assigned. A set of 180 approximate interproton distance constraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 33 phi constraints obtained for JNH alpha coupling data and the 23 psi dihedral angles identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Ten independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 30 calculated structures satisfy the experimental constraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 30 converged structures indicates that there are two helical regions extending from residues 4-16 and from residues 30-40, which are well defined both in terms of 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 2 A. The two helices exhibit typical amino acid preferences for specific locations at the ends of helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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].     

Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance

"Pseudo-structures" of the 20 common amino acid residues are introduced for use in protein spatial structure determinations, which rely on the use of intramolecular proton-proton distance constraints determined by nuclear Overhauser effects as input for distance geometry calculations. The proposed structures satisfy requirements for the initial structural interpretation of the nuclear magnetic resonance data that arise from the absence of stereospecific assignments and/or limited spectral resolution for certain resonance lines. The pseudo-atoms used as reference points for the experimental distance constraints can be used in conjunction with the real amino acid structures representing the van der Waals' constraints on the spatial molecular structure, or with simplified models in order to reduce the computing time for the distance geometry calculations.     

Conformation and dynamics of the three-helix bundle UBA domain of p62 from experiment and simulation

The ubiquitin associated domain of p62 is a small three-helix bundle of approximately 50 residues that mediates the recognition of polyubiquitin chains and ubiquitylated substrates. The solution structure of a 52 residue construct containing this domain has been characterized using heteronuclear nuclear magnetic resonance (NMR) methods. The resulting ensemble of NMR-derived structures was used in molecular dynamics (MD) simulations to investigate the equilibrium conformation and dynamics of this domain. NOE and (15)N relaxation data have been used to validate the structural ensemble produced by the MD simulations and show a good correlation for residues in regions of secondary structure. A similar approach was taken using an ensemble of structures from the MD simulations to calculate electronic circular dichroism (CD) and IR spectra from first principles with an encouraging correlation with the experimental CD and IR data.     

Three-dimensional structure of the wild-type lac Pribnow promoter DNA in solution. Two-dimensional nuclear magnetic resonance studies and distance geometry calculations

The solution structure of a 12 base-pair DNA duplex containing the wt-lac promoter Pribnow sequence TATGTT has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Proton assignments for the 24 sugar and base residues were obtained from two-dimensional correlated nuclear magnetic resonance and two-dimensional nuclear Overhauser effect spectra in both 2H2O and H2O, and by two-dimensional relayed coherence transfer nuclear magnetic resonance spectroscopy experiments. Time-dependent, two-dimensional nuclear Overhauser effect spectra were used to determine the initial cross-relaxation rates between 212 pairs of assigned protons, leading to 212 interproton distances in the double helix (8 to 9 per nucleotide). These distance constraints, and known bond lengths and angles, were entered into a distance matrix. After smoothing the bounds of the distance matrix, 12 trial matrices within the bounds constraints were independently generated and embedded in three-dimensional space using a distance geometry algorithm, to generate 12 trial structures. These trial structures were then refined until they no longer violated the distance matrix. The resulting structures are very similar at the local base-pair and nearest-neighbor base-pair level, but exhibit increasing variation at more distant and global levels. At the nearest-neighbor level, the A to T step and the G to T step within the Pribnow hexamer, as well as the G to T step preceding the hexamer, all exhibit very low screw pitch, i.e. 5(+/- 6) degrees. Conversely, the T to G step in the center of the promoter has a large screw pitch (47(+/- 2) degrees) and the T to G step at the 3' end of the promoter has a very large screw pitch (60(+/- 3) degrees). The limitations of nuclear magnetic resonance spectroscopy distance determination of structure are discussed in terms of resolution and spectral overlap of two-dimensional nuclear Overhauser effect crosspeaks. In the present duplex, the inability to measure several 1'-2' and 1'-2" distances resulted in underdetermination of the precise local sugar conformation for seven of the 24 residues, although the spatial position of all sugars was well defined.     

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Determining the three-dimensional fold of a protein from approximate constraints

We propose a new approach for calculating the three-dimensional (3D) structure of a protein from distance and dihedral angle constraints derived from experimental data. We suggest that such constraints can be obtained from experiments such as tritium planigraphy, chemical or enzymatic cleavage of the polypeptide chain, paramagnetic perturbation of nuclear magnetic resonance (NMR) spectra, measurement of hydrogen-exchange rates, mutational studies, mass spectrometry, and electron paramagnetic resonance. These can be supplemented with constraints from theoretical prediction of secondary structures and of buried/exposed residues. We report here distance geometry calculations to generate the structures of a test protein Staphylococcal nuclease (STN), and the HIV-1 rev protein (REV) of unknown structure. From the available 3D atomic coordinates of STN, we set up simulated data sets consisting of varying number and quality of constraints, and used our group's Self Correcting Distance Geometry (SECODG) program DIAMOD to generate structures. We could generate the correct tertiary fold from qualitative (approximate) as well as precise distance constraints. The root mean square deviations of backbone atoms from the native structure were in the range of 2.0 A to 8.3 A, depending on the number of constraints used. We could also generate the correct fold starting from a subset of atoms that are on the surface and those that are buried. When we used data sets containing a small fraction of incorrect distance constraints, the SECODG technique was able to detect and correct them. In the case of REV, we used a combination of constraints obtained from mutagenic data and structure predictions. DIAMOD generated helix-loop-helix models, which, after four self-correcting cycles, populated one family exclusively. The features of the energy-minimized model are consistent with the available data on REV-RNA interaction. Our method could thus be an attractive alternative for calculating protein 3D structures, especially in cases where the traditional methods of X-ray crystallography and multidimensional NMR spectroscopy have been unsuccessful.     

Supramolecular structure in full-length Alzheimer's beta-amyloid fibrils: evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance

We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (A beta(1-40)) obtained from solid-state nuclear magnetic resonance (NMR) measurements of intermolecular dipole-dipole couplings between (13)C labels at 11 carbon sites in residues 2 through 39. The measurements are carried out under magic-angle spinning conditions, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) technique. We also present one-dimensional (13)C magic-angle spinning NMR spectra of the labeled A beta(1-40) samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8 +/- 0.5 A for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly beta-sheet structure of the amyloid fibrils. The one-dimensional NMR spectra indicate structural order at these sites. The fpRFDR-CT data and NMR spectra also indicate structural disorder in the N-terminal segment of A beta(1-40), including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length beta-amyloid fibrils.     

Nuclear magnetic resonance determination of metal-proton distances in a synthetic calcium binding site of rabbit skeletal troponin C

The binding of gadolinium to a synthetic peptide of 13 amino acid residues representing the calcium binding loop of site 3 of rabbit skeletal troponin C [AcSTnC(103-115)amide] has been studied by using proton nuclear magnetic resonance (1H NMR) spectroscopy. In particular, the proton line broadening and enhanced spin-lattice relaxation have been used to determine proton-metal ion distances for several assigned nuclei in the peptide-metal ion complex. These distances have been used in conjunction with other constraints and a distance algorithm procedure to demonstrate that the structure of the peptide-metal complex as shown by 1H NMR is consistent with the structure of the EF calcium binding loop in the X-ray structure of parvalbumin but that the available 1H NMR distances do not uniquely define the solution structure.     

Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy

With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.     

A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular1H−1H proximities in solution

DISGEO is a new implementation of a distance geometry algorithm which has been specialized for the calculation of macromolecular conformation from distance measurements obtained by two-dimensional nuclear Overhauser enhancement spectroscopy. The improvements include (1) a decomposition of the complete embedding process into two successive, more tractable calculations by the use of “substructures”, (2) a compact data structure for storing incomplete distance information on a structure, (3) a more efficient shortest-path algorithm for computing the triangle inequality limits on all distances from this information, (4) a new algorithm for selecting random metric spaces from within these limits, (5) the use of chirality constraints to obtain good covalent geometry without the use ofad hoc weights or excessive optimization. The utility of the resultant program with nuclear magnetic resonance data is demonstrated by embedding complete spatial structures for the protein basic pancreatic trypsin inhibitor vs all 508 intramolecular, interresidue proton-proton contacts shorter than 4.0 Å that were present in its crystal structure. The crystal structure could be reproduced from this data set to within 1.3 Å minimum root mean square coordinate difference between the backbone atoms. We conclude that the information potentially available from nuclear magnetic resonance experiments in solution is sufficient to define the spatial structure of small proteins.     

Nuclear magnetic resonance solution structure of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis).

The three-dimensional structure in solution of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis) has been determined by nuclear magnetic resonance spectroscopy. A high quality structure for this 60-residue protein was obtained from 656 NOE distance constraints and 143 dihedral angle constraints, using the distance geometry program DIANA for the structure calculation and AMBER for restrained energy minimization. For a group of 20 conformers used to represent the solution structure, the average root-mean-square deviation value calculated for the polypeptide backbone heavy atoms relative to the mean structure was 0.45 A. The protein consists of a core region from which three finger-like loops extend outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 1-5 and 13-17, a three-stranded antiparallel beta-sheet involving residues 23-31, 34-42 and 51-55, and four disulfide bridges in the core region. There is also extensive non-regular hydrogen bonding between the carboxy-terminal tail of the polypeptide chain and the rest of the core region. Comparison with the crystal structure of erabutoxin-b indicates that the structure of alpha-neurotoxin is quite similar to other neurotoxin structures, but that local structural differences are seen in regions thought to be important for binding of neurotoxins to the acetylcholine receptor. For two regions of the alpha-neurotoxin structure there is evidence for an equilibrium between multiple conformations, which might be related to conformational rearrangements upon binding to the receptor. Overall, the alpha-neurotoxin presents itself as a protein with a stable core and flexible surface areas that interact with the acetylcholine receptor in such a way that high affinity binding is achieved by conformational rearrangements of the deformable regions of the neurotoxin structure.     

Solution conformation on bovine growth hormone releasing factor by 1H NMR and molecular modeling.

The structure of bovine growth hormone releasing factor (bGHRF) consisting of 44 amino acids has been studied in CD and 1H nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular modeling. Since bGHRF does not have an ordered structure in water alone, a 30% 2,2,2-trifluoroethanol (TFE) aqueous solvent was used to induce considerable alpha-helical structures, which corresponds to a helical content of approximately 62% as determined by circular dichroism (CD). The secondary structure was obtained from nuclear Overhauser enhancement and 3J(HN alpha) coupling constant in 30% TFE solution. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculation. A set of 267 interproton distances derived from nuclear Overhauser effect correlation spectroscopy (NOESY) experiments and coupling constants were used. From the initial random conformations, 50 distance geometry structures with minimal violations were selected for further refinement. The 14 best structures were obtained after simulated annealing calculation with energy minimization. The structure of bGHRF in 30% TFE solution was characterized by one alpha-helix (residues 8-19), two poorly constrained helices (residues 23-27 and residues 31-34) and a beta I(III)-turn fragment (residues 20-23; phi(i+1) = -53.1 degrees, psi(i+1) = -19.6 degrees, phi(i+2) = -59.9 degrees, psi(i+2) = -20.6 degrees) connected by the segments of less defined structures in N-terminal and omega-shaped flexible C-terminal determined from NOESY cross peaks between helical segment (residues 14-18) and tail fragment (residues 42-44). The obtained structure will play an important role toward the understanding of the structural and functional role of the GHRF.     

Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations

The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.     

Human interleukin 4. The solution structure of a four-helix bundle protein.

Heteronuclear 13C and 15N three-dimensional nuclear magnetic resonance (n.m.r.) techniques have been used to determine the solution structure of human interleukin 4, a four-helix bundle protein. A dynamical simulated annealing protocol was used to calculate an ensemble of structures from an n.m.r. data set of 1735 distance restraints, 101 phi angle restraints and 27 pairs of hydrogen bond restraints. The protein structure has a left-handed up-up-down-down topology for the four helices with the two long overhand loops in the structure being connected by a short section of irregular antiparallel beta-sheet. Analysis of the side-chains in the protein shows a clustering of hydrophobic residues, particularly leucines, in the core of the bundle with the side-chains of charged residues being located on the protein surface. The solution structure has been compared with a recent structure prediction for human interleukin 4 and with crystal structures of other helix bundle proteins.     

Structure of the 1-36 N-terminal fragment of human phospholamban phosphorylated at Ser-16 and Thr-17

The structure of a 36-amino-acid-long N-terminal fragment of human phospholamban phosphorylated at Ser-16 and Thr-17 and Cys-36-->Ser mutated was determined from nuclear magnetic resonance data in aqueous solution containing 30% trifluoroethanol. The peptide assumes a conformation characterized by two alpha-helices connected by an irregular strand, which comprises the amino acids from Arg-13 to Pro-21. The proline is in a trans conformation. The two phosphate groups on Ser-16 and Thr-17 are shown to interact preferably with the side chains of Arg-14 and Arg-13, respectively. The helix comprising amino acids 22 to 35 is well determined (the rmsd for the backbone atoms, calculated for a family of 24 nuclear magnetic resonance structures is 0.69 +/- 0.28 A). The structures of phosphorylated and unphosphorylated phospholamban are compared, and the effect of the two phosphate groups on the relative spatial position of the two helices is examined. The packing parameters Omega (interhelical angle) and d (minimal interhelical distance) are calculated: in the case of the phosphorylated phospholamban, Omega = 100 +/- 35 degrees and d = 7.9 +/- 4.6 A, whereas for the unphosphorylated peptide the values are Omega = 80 +/- 20 degrees and d = 7.0 +/- 4.0 A. We conclude that 1) the phosphorylation does not affect the structure of the C terminus between residues 21 and 36 and 2) the phosphorylated phospholamban has more loose helical packing than the nonphosphorylated.     

Solution structure of human growth hormone releasing factor. Combined use of circular dichroism and nuclear magnetic resonance spectroscopy

The solution structures of two human growth hormone releasing factor analogues, 27Leu45Gly-hGHRF(1-45)OH and 27Nle-hGHRF(1-29)NH2, are investigated by means of circular dichroism and nuclear magnetic resonance spectroscopy. Using circular dichroism spectroscopy, it is shown that both peptides adopt ordered structures at low concentrations of trifluoroethanol (approximately 30%). Quantitative analysis of the circular dichroism spectra indicates that the same number of residues, approximately 23 to 25, are in a helical state in both peptides. Using two-dimensional nuclear magnetic resonance methods all proton resonances of the 27Nle-hGHRF(1-29)NH2 fragment are assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. Two distinctive regions of alpha-helix are present extending from residues 6 to 13 and 16 to 29.