Turn-like Structures Research Articles

Secondary structure determination of 15N-labelled human Long-[Arg-3]-insulin-like growth factor 1 by multidimensional NMR spectroscopy

Insulin-like growth factors (IGFs) are a group of proteins that promote cell growth and differentiation. Long-[Arg-3]-IGF-I (Francis et al. (1992) J. Mol. Endocrinol. 8, 213–223), a potent analogue of IGF-I, which has a Glu-3 to Arg-3 substitution and a hydrophobic, thirteen amino acid N-terminal extension, has been studied by 1H, 15N NMR spectroscopy. All the backbone 1H and 15N assignments and most of the 1H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity-enhanced 15N-NOESY-HSQC and sensitivity-enhanced 15N-HSQC-NOESY-HSQC spectra. The IGF-I domain of Long-[Arg-3]-IGF-I was found to have an almost identical structure to IGF-I. The N-terminal seven amino acid residues of the extension have very few medium range or long range NOEs but the next five amino acids form a turn-like structure that is spatially close to the beginning of helix 1 in the IGF-I domain. Hydrogen-deuterium exchange experiments show that all the slowly exchanging backbone amide protons in the IGF-I domain are either in the helical or the extended structural elements. Many of the amide protons in the N-terminal extension are also protected from the solvent although the residues in this part of the extension do not have any identifiable secondary structure. The results are interpreted in terms of the increased biological potency of Long-[Arg-3]-IGF-I and the decreased binding to insulin-like growth factor binding proteins.

The bioactive conformation of neuropeptide Y analogues at the human Y2-receptor.

Several attempts to investigate the bioactive conformation of neuropeptide Y have been made so far. As cyclic peptides are much more rigid than linear ones, we decided to synthesise cyclic analogues of the C-terminal dodekapeptide amide neuropeptide Y Ac-25-36. Cyclisation was performed by side chain lactamisation of ornithine or lysine and glutamic or aspartic acid. The affinity of the 19 peptides ranged from Ki 0.6 nM to greater than 10,000 nM. We found that the size, position, orientation, configuration. and the location of the cycle plays an important role for receptor recognition. Circular dichroic studies have been performed to characterise the secondary structure of each peptide. Receptor binding studies were carried out on human neuroblastoma cell lines SK-N-MC (Y1) and SMS-KAN (Y2), and on rabbit kidney membranes (Y2). The pharmacological and spectral data showed that the alpha-helix content was not the predominant factor for high Y2-receptor affinity. Instead, the location and the size of the hydrophobic lactam bridge, and the conserved C-terminal tetrapeptide (Arg-Glu-Arg-Tyr) seemed to be the main parameters. Using molecular dynamics, the structures of four cyclic peptides (i,i+4) have been investigated and compared with the previously published NMR structure of one of the cyclic peptide analogues. Significant differences have been found in the overall three-dimensional fold of the peptides. The distances between the N- and the C-terminus allow discrimination between peptides with high binding affinity and those with low binding affinity, because of the correlation that was found with the measured affinity. Thus, this study suggests that a turn-like structure and the orientation of the C-terminus towards the N-terminus play major roles for high affinity binding of cyclic dodecapeptides to the Y2-receptor. None of the cyclic segments exhibits significant affinity to the Y1-receptor. Thus, these results support the hypothesis of a discontinuous binding site of neuropeptide Y at the Y1-receptor.

Conformation of 60-residue peptide fragment from N-terminal of porcine kidney fructose 1,6-bisphosphatase

Limited digestion of fructose 1, 6-bisphosphatase with subtilisin produces an S-peptide with an about 60-residue peptide fragment that is non-covalently associated with the enzyme. The 60-residue peptide fragment consists of the most part of allosteric site for AMP binding. It could be separated from S-protein by gel filtration with a Sephadex G-75 column equilibrated with 9% formic acid. According to X-ray diffraction results the S-peptide consists of two alpha-helices without beta-strand and the alpha-helix content is about 60% in the 60-residue-peptide fragment. When the enzyme is subjected to limited proteolysis with subtilisin, the secondary structure of the enzyme does not show a detectable change in CD spectrum. The CD spectra of the isolated S-peptide were measured under different concentrations. In the absence of GuHCl, S-peptide had 30% alpha-helix and 38.5% turn-like structure but had no beta-strand, suggesting that the N-terminal 60-residue fragment, which is synthesized initially by ribosome, would form a conformation spontaneously similar to that of the isolated 60-residue-peptide, i.e. about 30% alpha-helix and 30% turn-like structure. As the elongation of the peptide chain of the enzyme proceeds, the newly synthesized segment or the final entire enzyme, in turn, affects the conformation of prior peptide segment and adjusts its conformation to the final native state. The content of alpha-helix did not increase as perturbing the conformation of S-peptide by adding ethanol, cyclohexane or a small amount of SDS. On the contrary, the ordered structure was slightly decreased, indicating that the difference of conformations of S-peptide in the isolated form and in the associated protein was not an artifact produced by isolation process.

NMR studies of unfolded states of an SH3 domain in aqueous solution and denaturing conditions.

The isolated N-terminal SH3 domain of the Drosophila adapter protein drk (drkN SH3 domain) exists in a dynamic equilibrium between a folded (F(exch)) and an unfolded (U(exch)) state under native-like buffer conditions [Zhang, O., & Forman-Kay, J. D. (1995) Biochemistry 34, 6784-6794]. The effect of binding a proline-rich peptide derived from the protein Sos, a biological target of the drkN SH3 domain, on this equilibrium has been investigated. The stabilization of the F(exch) folded state upon binding provides an example of the link between binding and protein folding or stabilization. We have compared NMR parameters of the U(exch) state with those of a denatured state in 2 M guanidine hydrochloride (U(Gdn)). Variable-temperature experiments demonstrate that interactions in a region encompassing residues Gln 23-Leu 28 in the U(exch) state are destabilized upon addition of guanidine hydrochloride. Data from an 15N HSQC-NOESY-HSQC experiment as well as recently developed methods provide more unambiguous structural information than described previously, showing the presence of preferential structure in both unfolded states. Backbone NOEs observed in both unfolded states as well as chemical shifts and coupling constants suggest a rapid equilibrium between extended structure and turn-like structures which may play a role in initiation of protein folding. However, differences in detailed structural features between the two unfolded states argue that caution is needed in interpretation of results from structural characterization of protein conformational states generated using denaturing conditions.

Solution conformation of bovine lens alpha- and betaB2-crystallin terminal extensions.

alpha- and betaB2-Crystallin are the major proteins in the mammalian lens. Each of these crystallins has short, flexible terminal extensions from its domain core; the two alpha-crystallin subunits have C-terminal extensions of eight and ten amino acids whilst betaB2-crystallin has N- and C-terminal extensions of 15 and 11 amino acids, respectively. The solution conformations of these chemically synthesised extensions have been examined by two-dimensional 1H NMR spectroscopy. The N-terminal extension of betaB2-crystallin and the C-terminal extensions of alpha-crystallin adopt little ordered structure. In the membrane-mimicking solvent trifluoroethanol, the alpha-crystallin extensions are also unstructured. In contrast, the C-terminal extension of betaB2-crystallin in water has a structural preference towards turn-like structures, creating a hydrophobic region involving G198, F200 and P202. In the lens, the C-terminal extension of betaB2-crystallin is the only one of these extensions that interacts to any large extent with other crystallins. The structural preference of the C-terminal extension of betaB2-crystallin may therefore have implications for the role of this extension in crystallin-crystallin interactions.

Trimeric assembly and three-dimensional structure model of the FACIT collagen COL1-NC1 junction from CD and NMR analysis.

The 3D structure of the COL1-NC1 junction of FACIT type XIV collagen was investigated using GYCDPSSCAG and (GPP*)3GYCDPSSCAG synthetic peptides, circular dichroism, and NMR. At -20 degrees C and under air oxidation catalyzed by Cu2+, the peptide (GPP*)3GYCDPSSCAG is able to self-associated with high yield into a stable triple disulfide bonded trimer. The presence of a triple helical conformation was confirmed by circular dichroism. The analysis of the trimer by 2D NMR provided a set of distance constraints for the noncollagenous part. Molecular models for the 3D structure of COL1-NC1 junction were calculated, using the NMR distance constraints in combination with the 3D structural data recently established by X-ray crystallography [Bella, J., Eaton, M., Brodsky, B., & Berman, H. M. (1994) Science 266, 75-81] for a collagenous triple helix. From the eight theoretically possible arrangements for the three interchain disulfide bonds, only two close disulfide conformers are compatible with the experimental data. The main feature of the trimer structure is the asymmetry of the molecule due to the disulfide bond pattern that induces a particular folding of one chain. This chain forms a turn-like structure locked by two disulfide bonds with the two other chains. The turn-like folding is close to that observed for the cyclized oxidized monomeric peptide. This is the first report of the 3D structure model for a junction between a collagenous triple helical domain and a noncollagenous domain.

Aging of Dry Desiccation-Tolerant Pollen Does Not Affect Protein Secondary Structure.

Protein secondary structure and membrane phase behavior in aging Typha latifolia pollen were studied by means of Fourier transform infrared microspectroscopy (FTIR). Membranes isolated from fresh pollen occurred mainly in the liquid crystalline phase at room temperature, whereas the membrane fluidity of aged pollen was drastically decreased. This decrease did not result in large-scale irreversible protein aggregation, as was concluded from in situ FTIR assessment of the amide-1 bands. Curve-fitting on the infrared absorbance spectra enabled estimation of the proportion of different classes of protein secondary structure. Membrane proteins had a relatively large amount of [alpha]-helical structure (48%; band at 1658 cm-1), and turn-like structures (at 1637 and 1680 cm-1) were also detected. The secondary protein structure of isolated cytoplasmic proteins resembled that of proteins in whole pollen and was conserved upon drying in the absence of sucrose. The isolated cytoplasmic proteins had a large amount of [alpha]-helical structure (43%), and also [beta]-sheet (at 1637 and 1692 cm-1) and turn structures were detected. Heat-denaturing experiments with intact hydrated pollen showed low (1627 cm-1) and high (1692 cm-1) wave number bands indicating irreversible protein aggregates. The results presented in this paper show that FTIR is an extremely suitable technique to study protein secondary structure in intact plant cells of different hydration levels and developmental stages.

1H NMR Studies of Sarafotoxin SRTb, a Nonselective Endothelin Receptor Agonist, and IRL 1620, an ETB Receptor-Specific Agonist

1H NMR studies on the nonselective endothelin receptor agonist sarafotoxin SRTb have identified a helix between residues Asp 8 and His 16, and a beta-turn involving residues Cys 3 to Met 6; however, the biologically important C-terminal five residues were found to be conformationally variable. The average RMSD, measured for the final 43 refined structures to the average structure over residues 1-16, was 0.78 +/- 0.18 A for the backbone atoms and 1.39 +/- 0.22 A for all atoms. The torsion angles Cys 3 psi/Lys 4 theta, Thr 7 psi/Asp 8 theta and Gln 17 theta were identified as sites of conformational variability. Differences were found between the structures in the bicyclic loop region for SRTb and those published for ET1, another nonselective receptor agonist, which may explain the observed differences in potency of these peptides. The conformation of an ETB receptor-specific agonist, IRL 1620, which lacks the N-terminal seven residues and the two intrachain disulfides, was found by NMR and circular dichroism spectroscopy to be predominantly random coil, despite the fact that its affinity for the ETB receptor almost equals that of ET1. However, close analysis of the NMR results indicated the presence of turn-like structures, or a nascent helix, in the part of the sequence corresponding to the helical region in the parent peptides. These results suggest that the helical conformation may be required for ligand binding to the ETB receptor as well as to the ETA receptor.

Synthetic alpha-helical peptides incorporating intercalators for DNA recognition.

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz 1H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential dNN(i, i+1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an alpha-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.

Predicted conformation of poly(dehydroalanine): A preference for turns

Repetitive conformations of poly(dehydroalanine) were studied using molecular mechanics. An exhaustive search of the conformational space was carried out on a ΔAla octapeptide model, using the AMBER force field and the ΔAla parameters of Alagona et al [26], under three dielectric conditions, ϵ = 1 (vacuum), ϵ = r and ϵ = 4 r (solvent). In all cases, two major groups of low-energy conformers were found, one corresponding to a regular 3/10 helix or type III turn, the other to an irregular conformation, Φ = −157 to −170° Ψ = −1 to 15° which however can be found in the i + 2 position of gamma-turns. The data confirm that ΔAla may induce turn-like structures in peptides and also indicate that it may confer flexibility to the peptide chain. 0fa]This paper is dedicated to the memory of George Némethy, who recently deceased, in recognition of his contributions to this field, without which this work could not have been accomplished.

Three-dimensional structure of echistatin and dynamics of the active site.

The snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser effect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment from Gly5 to Cys20 and from Asp30 to Asn42 has a well-defined conformation and the Arg-Gly-Asp sequence, which adopts a turn-like structure, is located at the apex of a nine-residue loop connecting the two strands of a distorted beta-sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by 15N relaxation measurements. The overall correlation time of echistatin was determined from fluorescence measurements, and was used in a model-free analysis to determine internal motional parameters. The active site has order parameters of 0.3-0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution structures was made by calculating generalized order parameters from the ensemble of three-dimensional structures. The motion of the RGD site detected experimentally is more extensive than a simple RGD loop 'wagging' motional model, suggested by an examination of superposed solution structures.

Conformation of thymosin β4 in water determined by NMR spectroscopy

The conformational preferences of a 43-amino-acid G-actin-binding peptide, thymosin beta 4, in water at 1, 4 and 14 degrees C, and at pH 3.0 and 6.5 were studied by NMR. NMR showed that thymosin beta 4 lacks a uniquely folded conformation in water. However, some preferential alpha-helical conformations of thymosin beta 4 can be observed in aqueous solutions. The segment at residues 5-16 showed characteristic interactions for conformations in both the beta-strand and alpha-helical regions of the phi-psi space, based on strong C alpha H(i)-NH(i+1) interactions and NH-NH, C alpha H(i)-NH(i+3), and C alpha H(i)-C beta H(i+3) interactions, respectively. At 1-4 degrees C, another segment at residues 31-37 also shows both beta and alpha conformations, forming however a less well-defined helix than the segment at residues 5-16. At 14 degrees C, the conformational population of the helix at positions 5-16 is shifted more towards the random and turn-like structures, whereas the segment at positions 31-37 becomes exclusively a random coil.

A refined three-dimensional solution structure of a carboxy terminal fragment of apolipoprotein CII.

The three-dimensional structure of a synthetic fragment of human apolipoprotein CII (apo-CII) in 35%, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) has been determined on the basis of distance and intensity constraints derived from two-dimensional proton nuclear magnetic resonance measurements. The NOE crosspeak build-up rates were converted to distance constraints which were used in the distance geometry program DI-ANA. A set of one hundred structures were generated and of these ten structures were used in molecular dynamics simulations using the program XPLOR. This program enabled a direct minimization between the difference of the two-dimensional NOE intensities and those calculated from the full relaxation matrix. In this way spin diffusion is fully taken into account, which can be seen from the considerable improvement of the R-factor after the relaxation matrix refinement. These calculations show that this fragment, which corresponds to the carboxy terminal 30 amino acids of intact apo-CII and which retains its ability to activate lipoprotein lipase, is essentially flexible, but has three defined secondary structural elements. The most significant one is an alpha-helix between residues 67 and 74. The following three residues adopt a turn-like structure. Another turn of alpha-helix is seen between residues 56 and 59. The effect of the solvent system on the secondary structure was studied by circular dichroism spectroscopy. The results show that the mixed aqueous 35% HFP solvent induces secondary structure of a very similar nature to the one induced by sodium dodecyl sulphate.

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9 and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, beta-branched D-aliphatic residues at position 7 combined with bulky L-aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D-Arg0,Hyp3,Thi5,D-Cpg7,Cpg8]-BK [Cpg: alpha-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L-proline; Thi: beta-(2-thienyl)-L-alanine] (I), [D-Arg0,Hyp3,D-Cpg7,Cpg8]-BK (II), as well as its variant with D-Cpg7 replaced by Cpg7, namely [D-Arg0,Hyp3,Cpg7,Cpg8]-BK (III). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II, in direct correlation with the peptide activities. No turn-like structure was found for residues 6-9. In peptide III, a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists (I, II) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D-Cpg7]-BK (IV) showed no defined secondary structure.

Secondary Structure and Temperature-induced Unfolding and Refolding of Ribonuclease T1 in Aqueous Solution: A Fourier Transform Infrared Spectroscopic Study

Max-Delbrück-Centrum für Molekulare Medizin Robert-Rössle-Straße 10, D-1115 Berlin-Buch, Germany; Robert-Koch Institut des Bundesgesundheitsamtes Nordufer 20, D-1000 Berlin 65, Germany and Institut für Kristallographie der Freien Universität Berlin Takustraße 6, D-1000 Berlin 33, GermanyThe secondary structure of ribonuclease T1 (RNase T1) in aqueous solution and its temperature-induced structural changes have been investigated by Fourier-transform infrared (FT-IR) spectroscopy. 13 to 14% α-helix and 32 to 33% β-sheet were estimated from the resolution-enhanced FT-IR spectra, in agreement with the crystal structure which indicates 16% α-helix and 35% β-sheet. Specific IR-marker bands are assigned to the different β-sheet structures, to the slightly bent α-helix, and to β-turn and irregular conformations present in RNase T1. The temperature dependence of the infrared spectra shows that the thermal unfolding and refolding of RNase T1 is fully reversible. This permitted the detailed analysis of structural changes that occur as a function of temperature by evaluating quantitatively the various secondary structure-related amide I band components and some amino acid side-chain vibrations as specific monitors. The secondary structure of RNase T1 is essentially retained in the temperature range between 20 and 50°C. Significant perturbation of protein structure is initiated between 50 and 55°C within regions of β-sheet structures while the α-helix remains virtually intact up to 55°C suggesting a "premelting" of RNase T1. Between 55 and 60°C, a highly co-operative unfolding process is indicated by the simultaneous breakdown of all secondary structure components and by distinct changes of some specific side-chain vibrations. An analysis of the amide I band contour of RNase T1 at 70°C proves that the unfolded state is predominantly, but not completely, irregular or "random coil". Residual, turn-like structures persisting even in the unfolded state are suggested by minor, turn related band components in the amide I region. From IR-spectra collected along a linear temperature gradient, intensity/temperature and frequency/temperature profiles were constructed using some peptide backbone and amino acid side-chain marker bands as local, structure-sensitive monitors. From these profiles individual transition temperatures tm and transition enthalpies ΔH (van't Hoff) were calculated. The tm and ΔH values revealed a small but distinct hysteresis between repetitive cycles of unfolding and refolding of the protein, suggesting slow refolding kinetics of RNase T1. Furthermore, the various infrared "marker bands" indicate a slightly different response towards temperature increase/decrease for different regions of the protein. The data demonstrate that infrared spectroscopy permits both the detailed analysis of structural changes occurring in a protein as a function of temperature and the determination of thermodynamic parameters characterizing its folded/unfolded state transition.

Tendamistat (12–26) fragment

In order to determine whether regions of a protein that are turns in the native structure are able to maintain such a structure when isolated, we have studied the conformational properties of various peptide fragments corresponding to the 12-26-peptide region of the alpha-amylase inhibitor tendamistat, by NMR. Amide solvent accessibility, NOE spectroscopy (NOESY) and rotating-frame NOE spectroscopy (ROESY) data strongly support the conclusion that the 12-26 and 15-23 peptides adopt in aqueous solution, a set of turn-like structures located around the central region of their corresponding polypeptidic chains, the same region where a beta turn exists in the native protein. Such a set of structures are destabilized when one residue located within the native beta turn of the 15-23 peptide is modified Trp18----Ser. Our results indicate that the tendency to bend in a predetermined region of a protein chain seems to exist from the very beginning of the folding process and therefore it could drive the folding instead of being a consequence of the tertiary assembly of the protein.

Solution conformation of a peptide fragment representing a proposed RNA-binding site of a viral coat protein studied by two-dimensional NMR

The first 25 amino acids of the coat protein of cowpea chlorotic mottle virus are essential for binding the encapsidated RNA. Although an alpha-helical conformation has been predicted for this highly positively charged N-terminal region [Argos, P. (1981) Virology 110, 55-62; Vriend, G., Verduin, B. J. M., & Hemminga, M. A. (1986) J. Mol. Biol. 191, 453-460], no experimental evidence for this conformation has been presented so far. In this study, two-dimensional proton NMR experiments were performed on a chemically synthesized pentacosapeptide containing the first 25 amino acids of this coat protein [Ten Kortenaar, P. B. W., Krüse, J., Hemminga, M. A., & Tesser, G. I. (1986) Int. J. Pept. Protein Res. 27, 401-413]. All resonances could be assigned by a combined use of two-dimensional correlated spectroscopy and nuclear Overhauser enhancement spectroscopy carried out at four different temperatures. Various NMR parameters indicate the presence of a conformational ensemble consisting of helical structures rapidly converting into more extended states. Differences in chemical shifts and nuclear Overhauser effects indicate that lowering the temperature induces a shift of the dynamic equilibrium toward more helical structures. At 10 degrees C, a perceptible fraction of the conformational ensemble consists of structures with an alpha-helical conformation between residues 9 and 17, likely starting with a turnlike structure around Thr9 and Arg10. Both the conformation and the position of this helical region agree well with the secondary structure predictions mentioned above.

1H‐ and natural abundance 15N‐nmr studies of a derivative of a rabies glycoprotein fragment

Using a combination of one- and two-dimensional methods, 1H- and 15N-nmr spectroscopy has been employed to perform the complete assignment and the structural determination of the immunogenic undecapeptide CTTTNSRGTTT in DMSO solution. Nuclear Overhauser enhancement spectroscopy experiments indicated the presence of secondary structures, mainly turn-like structures, which only represent a family, albeit a dominant one, of an ensemble of conformations available to the peptide. Since reverse turns may play an important role as intermediates in protein folding, the experimental observations described here may link the immunological and theoretical approaches to protein folding.

Folding of immunogenic peptide fragments of proteins in water solution: II. The nascent helix

1H nuclear magnetic resonance experiments indicate formation of secondary structures in water solutions of a synthetic immunogenic peptide of sequence EVVPHKKMHKDFLEKIGGL corresponding to the C-helix (residues 69 to 87) of myohemerythrin. The conformational ensemble consists of a set of turn-like structures, distributed over the C-terminal half of the peptide and rapidly interconverting by way of unfolded states. These structures, termed nascent helix, are stabilized into helical structure with long-range order in water/trifluorethanol mixtures. Circular dichroism measurements confirm the presence of 50% helix in water/trifluoroethanol but show no evidence of helicity in water solutions of the peptide. It is apparent that no one member of the transient set of helical conformations which constitutes the nascent helix is sufficiently long to be detectable by circular dichroism experiments. No preferred conformations could be detected by nuclear magnetic resonance in the N-terminal half of the peptide, either in water or water/trifluoroethanol mixtures. This region of the peptide is stabilized in helix by long-range interactions in the folded protein. The possible role of nascent secondary structure in induction of antipeptide antibodies and in initiation of protein folding is discussed.