Toward direct determination of conformations of protein building units from multidimensional NMR experiments. V. NMR chemical shielding analysis of N-formyl-serinamide,a model for polar side-chain containing peptides

Knowledge of chemical shift-structure relationships could greatly facilitate the NMR chemical shift assignment and structure refinement processes that occur during peptide/protein structure determination via NMR spectroscopy. To determine whether such correlations exist for polar side chain containing amino acid residues the serine dipeptide model, For-L-Ser-NH(2), was studied. Using the GIAO-RHF/6-31+G(d) and GIAO-RHF/TZ2P levels of theory the NMR chemical shifts of all hydrogen ((1)H(N), (1)H(alpha), (1)H(beta1), (1)H(beta2)), carbon ((13)C(alpha), (13)C(beta), (13)C') and nitrogen ((15)N) atoms have been computed for all 44 stable conformers of For-L-Ser-NH(2). An attempt was made to establish correlation between chemical shift of each nucleus and the major conformational variables (omega(0), phi, psi, omega(1), chi,(1) and chi(2)). At both levels of theory a linear correlation can be observed between (1)H(alpha)/phi, (13)C(alpha)/phi, and (13)C(alpha)/psi. These results indicate that the backbone and side-chain structures of For-L-Ser-NH(2) have a strong influence on its chemical shifts.     

Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G2FT NMR experiments

A suite of novel (5,3)D G2FT triple resonance NMR experiments encoding highly resolved 5D spectral information is presented for sequential resonance assignment of proteins exhibiting high chemical shift degeneracy. Efficient resonance assignment is achieved by separate joint sampling of (i) chemical shifts which solely serve to provide increased resolution and (ii) shifts which also provide sequential connectivities. In these G2FT experiments, two G-matrix transformations are employed. Peaks are resolved along a first GFT dimension at both Omega(15N) + Omega(13C') and Omega(15N) - Omega(13C'), or at Omega(15N) + Omega(13Calpha) and Omega(15N) - Omega(13Calpha), to break backbone 15N,1HN chemical shift degeneracy. Sequential connectivities are established along a second GFT dimension by measuring intraresidue and sequential correlations at 2Omega(13Calpha), Omega(13Calpha + 13Cbeta), and Omega(13Calpha - 13Cbeta), or at Omega(13Calpha + 1Halpha) and Omega(13Calpha - 1Halpha), to resolve 13Calpha/beta,1Halpha chemical shift degeneracy. It is demonstrated that longitudinal proton relaxation optimization of out-and-back implementations suitable for deuterated proteins and nonlinear data sampling combined with maximum entropy reconstruction further accelerate G2FT NMR data acquisition speed. As a result, the spectral information can be obtained within hours, so that (5,3)D G2FT experiments are viable options for high-throughput structure determination in structural genomics. Applications are presented for 17 kDa alpha-helical protein YqbG and 13.5 kDa protein rps24e, targets of the Northeast Structural Genomics consortium, as well as for 9 kDa protein Z-domain. The high resolving power of the G2FT NMR experiments makes them attractive choices to study alpha-helical globular/membrane or (partially) unfolded proteins, thus promising to pave the way for NMR-based structural genomics of membrane proteins.     

A theoretical case study of type I and type II beta-turns

NMR chemical shielding anisotropy tensors have been computed by employing a medium size basis set and the GIAO-DFT(B3LYP) formalism of electronic structure theory for all of the atoms of type I and type II beta-turn models. The models contain all possible combinations of the amino acid residues Gly, Ala, Val, and Ser, with all possible side-chain orientations where applicable in a dipeptide. The several hundred structures investigated contain either constrained or optimized phi, psi, and chi dihedral angles. A statistical analysis of the resulting large database was performed and multidimensional (2D and 3D) chemical-shift/chemical-shift plots were generated. The (1)H(alpha-13)C(alpha), (13)C(alpha-1)H(alpha-13)C(beta), and (13)C(alpha-1)H(alpha-13)C' 2D and 3D plots have the notable feature that the conformers clearly cluster in distinct regions. This allows straightforward identification of the backbone and side-chain conformations of the residues forming beta-turns. Chemical shift calculations on larger For-(L-Ala)(n)-NH(2) (n=4, 6, 8) models, containing a single type I or type II beta-turn, prove that the simple models employed are adequate. A limited number of chemical shift calculations performed at the highly correlated CCSD(T) level prove the adequacy of the computational method chosen. For all nuclei, statistically averaged theoretical and experimental shifts taken from the BioMagnetic Resonance Bank (BMRB) exhibit good correlation. These results confirm and extend our previous findings that chemical shift information from selected multiple-pulse NMR experiments could be employed directly to extract folding information for polypeptides and proteins.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments VI: chemical shift analysis of his to gain 3D structure and protonation state information

NMR--chemical shift structure correlations were investigated by using GIAO-RB3LYP/6-311++G(2d,2p) formalism. Geometries and chemical shifts (CSI values) of 103 different conformers of N'-formyl-L-histidinamide were determined including both neutral and charged protonation forms. Correlations between amino acid torsional angle values and chemical shifts were investigated for the first time for an aromatic and polar amino acid residue whose side chain may carry different charges. Linear correlation coefficients of a significant level were determined between chemical shifts and dihedral angles for CSI[(1)H(alpha)]/phi, CSI[(13)C(alpha)]/phi, and CSI[(13)C(alpha)]/psi. Protonation of the imidazole ring induces the upfield shift of CSI[(13)C(alpha)] for positively charged histidines and an opposite effect for the negative residue. We investigated the correspondence of theoretical and experimental (13)C(alpha), (13)C(beta), and (1)H(alpha) chemical shifts and the nine basic conformational building units characteristic for proteins. These three chemical shift values allow the identification of conformational building units at 80% accuracy. These results enable the prediction of additional regular secondary structural elements (e.g., polyProlineII, inverse gamma-turns) and loops beyond the assignment of chemical shifts to alpha-helices and beta-pleated sheets. Moreover, the location of the His residue can be further specified in a beta-sheet. It is possible to determine whether the appropriate residue is located at the middle or in a first/last beta-strand within a beta-sheet based on calculated CSI values. Thus, the attractive idea of establishing local residue specific backbone folding parameters in peptides and proteins by employing chemical shift information (e.g., (1)H(alpha) and (13)C(alpha)) obtained from selected heteronuclear correlation NMR experiments (e.g., 2D-HSQC) is reinforced.     

Determination of multiple torsion-angle constraints in U-(13)C,(15)N-labeled peptides: 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift NMR spectroscopy in rotating solids

We demonstrate constraint of peptide backbone and side-chain conformation with 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift, magic-angle spinning NMR experiments. In these experiments, polarization is transferred from (15)N[i] by ramped SPECIFIC cross polarization to the (13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1] resonances and evolves coherently under the correlated (1)H-(15)N and (1)H-(13)C dipolar couplings. The resulting set of frequency-labeled (15)N(1)H-(13)C(1)H dipolar spectra depend strongly upon the molecular torsion angles phi[i], chi1[i], and psi[i - 1]. To interpret the data with high precision, we considered the effects of weakly coupled protons and differential relaxation of proton coherences via an average Liouvillian theory formalism for multispin clusters and employed average Hamiltonian theory to describe the transfer of (15)N polarization to three coupled (13)C spins ((13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1]). Degeneracies in the conformational solution space were minimized by combining data from multiple (15)N(1)H-(13)C(1)H line shapes and analogous data from other 3D (1)H-(13)C(alpha)-(13)C(beta)-(1)H (chi1), (15)N-(13)C(alpha)-(13)C'-(15)N (psi), and (1)H-(15)N[i]-(15)N[i + 1]-(1)H (phi, psi) experiments. The method is demonstrated here with studies of the uniformly (13)C,(15)N-labeled solid tripeptide N-formyl-Met-Leu-Phe-OH, where the combined data constrains a total of eight torsion angles (three phi, three chi1, and two psi): phi(Met) = -146 degrees, psi(Met) = 159 degrees, chi1(Met) = -85 degrees, phi(Leu) = -90 degrees, psi(Leu) = -40 degrees, chi1(Leu) = -59 degrees, phi(Phe) = -166 degrees, and chi1(Phe) = 56 degrees. The high sensitivity and dynamic range of the 3D experiments and the data analysis methods provided here will permit immediate application to larger peptides and proteins when sufficient resolution is available in the (15)N-(13)C chemical shift correlation spectra.     

Dynamics of ligand binding from 13C NMR relaxation dispersion at natural abundance

We show that Carr-Purcell-Meiboom-Gill (CPMG) 13Calpha NMR relaxation dispersion measurements are a viable means for profiling mus-ms ligand dynamics involved in receptor binding. Critically, the dispersion is at natural 13C abundance; this matches typical pharmaceutical research settings in which ligand isotope-labeling is often impractical. The dispersion reveals ligand 13Calpha nuclei that experience mus-ms modulation of their chemical shifts due to binding. 13Calpha shifts are dominated by local torsion angles , psi, chi1; hence, these experiments identify flexible torsion angles that may assist complex formation. Since the experiments detect the ligand, they are viable even in the absence of a receptor structure. The mus-ms dynamic information gained helps establish flexibility-activity relationships. We apply these experiments to study the binding of a phospho-peptide substrate ligand to the peptidyl-prolyl isomerase Pin1.     

Tryptophan chemical shift in peptides and proteins: a solid state carbon-13 nuclear magnetic resonance spectroscopic and quantum chemical investigation

We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.     

Secondary structures of peptides and proteins via NMR chemical-shielding anisotropy (CSA) parameters

Complete nuclear magnetic resonance (NMR) chemical-shielding tensors, sigma, have been computed at different levels of density-functional theory (DFT), within the gauge-including atomic orbital (GIAO) formalism, for the atoms of the peptide model For-L-Ala-NH2 as a function of the backbone dihedral angles phi and psi by employing a dense grid of 10 degrees. A complete set of rigorously orthogonal symmetric tensor invariants, {sigma iso, rho, tau}, is introduced, where sigma iso is the usual isotropic chemical shielding, while the newly introduced rho and tau parameters describe the magnitude and the orientation/shape of the chemical-shielding anisotropy (CSA), respectively. The set {sigma iso, rho, tau} is unaffected by unitary transformations of the symmetric part of the shielding tensor. The mathematically and physically motivated {rho, tau} anisotropy pair is easily connected to more traditional shielding anisotropy measures, like span (Omega) and skew (kappa). The effectiveness of the different partitions of the CSA information in predicting conformations of peptides and proteins has been tested throughout the Ramachandran space by generating theoretical NMR anisotropy surfaces for our For-L-Ala-NH2 model. The CSA surfaces, including Omega(phi, psi), kappa(phi, psi), rho(phi, psi), and tau(phi, psi) are highly structured. Individually, none of these surfaces is able to distinguish unequivocally between the alpha-helix and beta-strand secondary structural types of proteins. However, two- and three-dimensional correlated plots, including Omega versus kappa, rho versus tau, and sigma iso versus rho versus tau, especially for 13Calpha, have considerable promise in distinguishing among all four of the major secondary structural elements.     

Chi(1) rotamer populations and angles of mobile surface side chains are accurately predicted by a torsion angle database potential of mean force

The equilibrium angles and distributions of chi(1) rotamers for mobile surface side chains of the small, 63-residue, B1 domain of protein L have been calculated from the static crystal structure by rigid body/torsion angle simulated annealing using a torsion angle database potential of mean force and compared to those deduced by Monte Carlo analysis of side chain residual dipolar couplings measured in solution. Good agreement between theory and experiment is observed, indicating that for side chains undergoing rotamer averaging that is fast on the chemical shift time scale, the equilibrium angles and distribution of chi(1) rotamers are largely determined by the backbone phi/psi torsion angles.     

Quantum mechanical calculations of conformationally relevant 1H and 13C NMR chemical shifts of calixarene systems

[graphs: see text] QM GIAO calculations of 13C and 1H chemical shift values of the ArCH2Ar group have been performed, using the hybrid DFT functional MPW1PW91 and the 6-31G(d,p) basis set, on some representative calixarenes and on a series of simplified calixarene models allowing derivation of chemical shift surfaces versus phi and chi dihedral angles. A good reproduction of experimental data was obtained. The applicability of chemical shift surfaces in the study of calixarene conformational features is illustrated.     

Protein Backbone 1H(N)-13Calpha and 15N-13Calpha residual dipolar and J couplings: new constraints for NMR structure determination

A simple, sensitivity-enhanced experiment was devised for accurate measurement of backbone 15N-13Calpha and 1HN-13Calpha couplings in proteins. The measured residual dipolar couplings 2DHCA, 1DNCA, 3DHCA, and 2DNCA for protein GB1 display very good agreement with the refined NMR structure (PDB code: 3GB1). A Karplus-type relationship between the one-bond 1JNCA couplings and the backbone dihedral psi angles holds, and on the basis of the two-bond 2JNCA couplings a secondary structure index can be established.     

Solid-state NMR spectroscopy method for determination of the backbone torsion angle psi in peptides with isolated uniformly labeled residues

We demonstrate a solid-state nuclear magnetic resonance technique, with the acronym ROCSA-LG, for the determination of backbone torsion angles psi in peptides with multiple, but isolated, uniformly labeled residues. The method correlates the 13C' chemical shift anisotropy and the 13Calpha-1Halpha heteronuclear dipolar tensors within a single uniformly labeled residue in a two-dimensional (2D) experiment. The technique requires the measurement of only five 2D spectra and is compatible with high-speed magic-angle spinning. Experimental results are presented for the 17-residue alpha-helical peptide MB(i+4)EK and for amyloid fibrils formed by the 15-residue peptide Abeta11-25.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments I. A theoretical case study of For‐Gly‐NH2 and For‐L‐Ala‐NH2

NMR chemical shielding anisotropy tensors have been computed, employing several basis sets and the GIAO‐RHF and GIAO‐MP2 formalisms of electronic structure theory, for all the atoms of the five and nine typical backbone conformers of For‐Gly‐NH₂ and For‐L ‐Ala‐NH₂, respectively. Multidimensional chemical shift plots, as a function of the respective backbone fold, have been generated for both peptide models. On the 2D ¹H^NH‐¹⁵N^NH and ¹⁵N^NH‐¹³C^α plots the most notable feature is that at all levels of theory studied the backbone conformers cluster in different regions. Computed chemical shifts, as well as their averages, have been compared to relevant experimental values taken from the BioMagnetic Resonance Bank (BMRB). At the highest levels of theory, for all nuclei but the amide protons, deviations between statistically averaged theoretical and experimental shifts are as low as 1–3%. These results indicate that chemical shift information from selected multiple‐pulse NMR experiments (e.g., 2D‐HSQC and 3D‐HNCA) could directly be employed to extract folding information for polypeptides and proteins. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 882–900, 2000     

Conformational analysis of the eight-membered ring of the oxidized cysteinyl-cysteine unit implicated in nicotinic acetylcholine receptor ligand recognition.

Nicotinic acetylcholine receptors (nAChRs) are membrane-bound, pentameric ligand-gated ion channels associated with a variety of human disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia, and pain. Most known nAChRs contain an unusual eight-membered disulfide-containing cysteinyl-cysteine ring, ox-[Cys-Cys], as does the soluble acetylcholine binding protein (AChBP) found in the snail Lymnaea stagnalis. The cysteinyl-cysteine ring is located in a region implicated in ligand binding, and conformational changes involving this ring may be important for modulation of nAChR function. We have studied the preferred conformations of Ac-ox-[Cys-Cys]-NH2 by NMR in water and computationally by Monte Carlo simulations using the OPLS-AA force field and GB/SA water model. ox-[Cys-Cys] adopts four distinct low-energy conformers at slightly above 0 degrees C in water. Two populations are dependent on the peptide omega2 dihedral angle, with the trans amide favored over the cis amide by a ratio of ca. 60:40. Two ox-[Cys-Cys] conformers with a cis amide bond (C+ and C-) differ from each other primarily by variation of the chi3 dihedral angle, which defines the orientation of the helicity about the S-S bond (+/- 90 degrees ). Two trans amide conformers have the same S-S helicity (chi3 approximately -90 degrees ), but are distinguished by a backbone rotation about phi2 and psi1 (T- and T'-). The ratio of T-/T'-/C+/C- is 47:15:29:9. The orientation of the pendant moieties from the eight-membered ring is more compact for the major trans conformer (T-) than for the extended conformations adopted by T'-, C+, and C-. These conformational preferences are also observed in tetrapeptide and undecapeptide fragments of the human alpha7 subtype of the nAChR that contains the ox-[Cys-Cys] unit. Conformer T- is nearly identical to the conformation seen in the X-ray structure of ox-[Cys(187)-Cys(188)] found in the unliganded AChBP, and is a Type VIII beta-turn.     

Microenvironmental and conformational structure of triblock copolymers in aqueous solution by 1H and 13C NMR spectroscopy

1H and 13C nuclear magnetic resonance (NMR) spectra of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers in D2O solutions have been systematically investigated. The detailed assignments of various 1H and 13C NMR signals are presented. The hyperfine structure of PO -CH2- protons was clearly assigned, the arising reason of this hyperfine structure was attributed to the influence of the chiral center of -CHCH3- groups and the direct coupling between the PO -CH2- and -CH3 protons. The external standard 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) was firstly applied in this system. Accurate chemical shift values referenced to the external standard DSS were obtained. 1H NMR chemical shift of PO -CH2- and -CH3 signals shows a larger decrease in ppm values than that of EO -CH2- signal with the increase of PPO/PEO ratio or temperature indicating that PO segments exist in a more hydrophobic microenvironment. A new resonance signal assigned to the PO -CH2- protons appeared when the temperature is above the CMT, which is attributed to the breakdown of the intra-molecular (C-H)...O hydrogen bond between the PO -CH2- protons and the ester oxygens. The breakdown of this intra-molecular hydrogen bond may result in a decrease of gauche conformers of the PPO chain. The increase of 13C NMR chemical shift of block copolymers validates this conformational change assumption. It can be inferred that the amount of gauche conformers decreases whereas that of trans conformers increases in both PO and EO chains when elevating the PPO/PEO ratio or temperature. The observed 13C NMR chemical shifts of PO segments show a bigger increase than those of EO segments, supporting the formation of a nonpolar microenvironment around PO segments.     

Quantum mechanical calculations of conformationally relevant 1H and 13C NMR chemical shifts of N-, O-, and S-substituted calixarene systems

QM GIAO calculations of (13)C and (1)H chemical shift values of the ArCH(2)Ar group in N-, O-, and S-substituted calixarene systems were performed with a hybrid DFT functional MPW1PW91 and 6-31G(d,p) basis set. A good reproduction of experimental data was obtained for some representative calixarenes and for a series of simplified calixarene models. This allowed the derivation of chemical shift surfaces versus phi and chi dihedral angles. The applicability of chemical shift surfaces in the study of calixarene conformational features is illustrated.     

Conformational flexibility of a microcrystalline globular protein: order parameters by solid-state NMR spectroscopy

The majority of protein structures are determined in the crystalline state, yet few methods exist for the characterization of dynamics for crystalline biomolecules. Solid-state NMR can be used to probe detailed dynamic information in crystalline biomolecules. Recent advances in high-resolution solid-state NMR have enabled the site-specific assignment of (13)C and (15)N nuclei in proteins. With the use of multidimensional separated-local-field experiments, we report the backbone and side chain conformational dynamics of ubiquitin, a globular microcrystalline protein. The measurements of molecular conformational order parameters are based on heteronuclear dipolar couplings, and they are correlated to assigned chemical shifts, to obtain a global perspective on the sub-microsecond dynamics in microcrystalline ubiquitin. A total of 38 Calpha, 35 Cbeta and multiple side chain unique order parameters are collected, and they reveal the high mobility of ubiquitin in the microcrystalline state. In general the side chains show elevated motion in comparison with the backbone sites. The data are compared to solution NMR order parameter measurements on ubiquitin. The SSNMR measurements are sensitive to motions on a broader time scale (low microsecond and faster) than solution NMR measurements (low nanosecond and faster), and the SSNMR order parameters are generally lower than the corresponding solution values. Unlike solution NMR relaxation-based order parameters, order parameters for (13)C(1)H(2) spin systems are readily measured from the powder line shape data. These results illustrate the potential for detailed, extensive, and site-specific dynamic studies of biopolymers by solid-state NMR.     

Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods

Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.     

Poly-L-proline type II peptide mimics based on the 3-azabicyclo[3.1.0]hexane system.

This paper describes conformational studies of proline-templated amino acids (PTAAs) based on the 3-azabicyclo[3.1.0]hexane system as well as conformational studies on short peptides composed of these PTAAs. NOE data, coupling constants, and molecular modeling are consistent with a flattened boat conformation for monomeric and oligomeric residues based on this bicyclic system. NMR studies on dimeric and trimeric oligomers are consistent with a populated poly-L-proline type II conformation in CDCl3 and D2O. Solution studies and molecular modeling predicts phi approximately -70 degrees, psi approximately 131 degrees, chi 1 approximately -57 degrees, and chi 2 approximately -158 degrees for oligomeric residues.     

Molecular carpentry: piecing together helices and hairpins in designed peptides

The design of a peptide that contains two distinct elements of secondary structure, helix and beta-hairpin, is described. Two designed 17-residue peptides: Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Gly-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe (I) and Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Gly-Gly-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe (II) have been conformationally characterized by NMR spectroscopy. Peptides I and II contain a seven-residue helical module at the N terminus and a eight-residue beta-hairpin module at the C terminus, which are connected by a conformationally flexible Gly-Gly segment. The choice of the secondary-structure modules is based upon prior crystallographic and spectroscopic analysis of the individual modules. Analysis of 500 MHz 1H NMR data, recorded as solutions in methanol, suggests that the observed pattern of chemical shifts, 3JHN CalphaH values, temperature coefficients of the NH chemical shifts, and backbone inter-residue nuclear Overhauser effects favor helical structures for residues 1-7 and beta-hairpin structures for residues 10-17. The spectroscopic data are compatible with termination of the helical segment by formation of a Schellman motif; this restricts Gly(8) to a left-handed alpha-helical conformation. Gly(9) is the only residue with multiple conformational possibilities in phi,psi space. Possible orientations of the two secondary-structure modules are considered. This study validates the use of stereochemically rigid peptide modules as prefabricated elements in the construction of synthetic protein mimics.