Conformational transitions in a Pro-Pro peptide on dissolution of single crystals

The peptide t-butyloxycarbonyl-α-aminoisobutyryl-L-prolyl-L-prolyl-N-methylamide has been shown to adopt an extended structure in the solid state. The Pro-Pro segment occurs in the poly-proline II conformation. On dissolution of single crystals at not, vert, similar 233°K, a single species corresponding to the all Image peptide backbone is observed by 270 MHz 1H NMR. On warming, Image to Image isomerization about the Pro-Pro bond is facilitated. Both Image (ψ not, vert, similar−50°) and Image (ψ not, vert, similar 130°) rotamers about the Pro3 Cα---CO bond are detectable in the Pro-Pro Image conformer, at low temperature. These observations demonstrate unambiguously the large differences in the solid state and solution conformations of a Pro-Pro sequence.

Cyclic Peptide Disulfides - Solution And Solid-State Conformation Of Boc-Cys-Pro-Aib-Cys-S-S-Bridge-Nhme,A Disulfide-Bridged Peptide Helix

The solution and solid-state conformations of the peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe have been determined by NMR spectroscopy and X-ray diffraction. The Cys(4) and methylamide NH groups are solvent shielded in CDCI3 and (CD,),SO, suggesting their involvement in intramolecular hydrogen bonding. On the basis of known stereochemical preferences of Pro and Aib residues, a consecutive @-turn structure is favored in solution. X-ray diffraction analysis reveals a highly folded 310 helical conformation for the peptide, with the S-S bridge lying approximately parallel to the helix axis, linking residues 1 and 4. The backbone conformational angles are Cys(1) 4 = -121.1', $ = 65.6"; Pro(2) 4 = -58.9', 4 = -34.0'; Aib(3) 4 = -61.8', $ = -17.9'; Cys(4) 4 = -70.5', $ = -18.6'. Two intramolecular hydrogen bonds are observed between Cys(1) CO--HN Cys(4) and Pro(2) CO--HNMe. The disulfide bond has a right-handed chirality, with a dihedral angle (xss) of 82'.

Parallel packing of alpha-helices in crystals of the zervamicin IIA analog Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe.2H2O.

An apolar synthetic analog of the first 10 residues at the NH2-terminal end of zervamicin IIA crystallizes in the triclinic space group P1 with cell dimensions a = 10.206 +/- 0.002 A, b = 12.244 +/- 0.002 A, c = 15.049 +/- 0.002 A, alpha = 93.94 +/- 0.01 degrees, beta = 95.10 +/- 0.01 degrees, gamma = 104.56 +/- 0.01 degrees, Z = 1, C60H97N11O13 X 2H2O. Despite the relatively few alpha-aminoisobutyric acid residues, the peptide maintains a helical form. The first intrahelical hydrogen bond is of the 3(10) type between N(3) and O(0), followed by five alpha-helix-type hydrogen bonds. Solution 1H NMR studies in chloroform also favor a helical conformation, with seven solvent-shielded NH groups. Continuous columns are formed by head-to-tail hydrogen bonds between the helical molecules along the helix axis. The absence of polar side chains precludes any lateral hydrogen bonds. Since the peptide crystallizes with one molecule in a triclinic space group, aggregation of the helical columns must necessarily be parallel rather than antiparallel. The packing of the columns is rather inefficient, as indicated by very few good van der Waals' contacts and the occurrence of voids between the molecules.

CSSI-PRO: a method for secondary structure type editing, assignment and estimation in proteins using linear combination of backbone chemical shifts

Estimation of secondary structure in polypeptides is important for studying their structure, folding and dynamics. In NMR spectroscopy, such information is generally obtained after sequence specific resonance assignments are completed. We present here a new methodology for assignment of secondary structure type to spin systems in proteins directly from NMR spectra, without prior knowledge of resonance assignments. The methodology, named Combination of Shifts for Secondary Structure Identification in Proteins (CSSI-PRO), involves detection of specific linear combination of backbone H-1(alpha) and C-13' chemical shifts in a two-dimensional (2D) NMR experiment based on G-matrix Fourier transform (GFT) NMR spectroscopy. Such linear combinations of shifts facilitate editing of residues belonging to alpha-helical/beta-strand regions into distinct spectral regions nearly independent of the amino acid type, thereby allowing the estimation of overall secondary structure content of the protein. Comparison of the predicted secondary structure content with those estimated based on their respective 3D structures and/or the method of Chemical Shift Index for 237 proteins gives a correlation of more than 90% and an overall rmsd of 7.0%, which is comparable to other biophysical techniques used for structural characterization of proteins. Taken together, this methodology has a wide range of applications in NMR spectroscopy such as rapid protein structure determination, monitoring conformational changes in protein-folding/ligand-binding studies and automated resonance assignment.

Acyclic peptides as conformational models Crystal structure of Boc-Aib-Leu-Pro-NHMe± 2H2O

The tripeptide Boc-Aib-Leu-Pro-NHMe crystallizes in the orthorhombic space group P212121 with a = 9.542, b = 15.200, c = 18.256 Å and Z = 4. Each peptide is associated wth two water molecules in the asymmetric unit of the crystal. The structure has been solved by direct methods and refined to an R-value of 0.069. The peptide adopts a structure without any intramolecular hydrogen bond. The three residues occupy distinctly different regions of the Ramachandran map: Aib in the left-handed 310-helical region (± = 67°, ± = 23°), Leu in the β-sheet region (± = - 133°, ± = 142°) and Pro in the poly (Pro) II region (± = - 69°, ± = 151°). An interesting observation is that each water molecule participates in four hydrogen bonds with distorted tetrahedral coordination about the oxygen atom.

X-Pro peptides. A theoretical study of the hydrogen bonded conformations of (alpha-aminoisobutyryl-L -prolyl)n sequences

Intramolecularly hydrogen bonded conformations of (Aib-Pro)n sequences have been analysed theoretically. Both 4-1 (C10 and 3-1 (C7 hydrogen bonded regular structures are shown to be stereochemically feasible. Conformational energies for the helical structures have been estimated using classical potential energy methods. Both C10 and C7 conformations have very similar energies. Pyrrolidine ring puckering has a pronounced effect on the energies, and only Cv-endo puckered Pro residues can be accommodated. The theoretical calculations using spectroscopic data suggest that the recently proposed novel 310 helical conformation for benzyloxycarbonyl(Aib-Pro)4-methyl ester is in solution, is indeed energetically and stereochemically favourable.

X-Pro peptides. A theoretical study of the hydrogen bonded conformations of (a-aminoisobutyryl-L -prolyl)n sequences

Intramolecularly hydrogen bonded conformations of (Aib-Pro)n sequences have been analysed theoretically. Both 4�1 (C10 and 3�1 (C7 hydrogen bonded regular structures are shown to be stereochemically feasible. Conformational energies for the helical structures have been estimated using classical potential energy methods. Both C10 and C7 conformations have very similar energies. Pyrrolidine ring puckering has a pronounced effect on the energies, and only Cγ-endo puckered Pro residues can be accommodated. The theoretical calculations using spectroscopic data suggest that the recently proposed novel 310 helical conformation for benzyloxycarbonyl(Aib-Pro)4-methyl ester is in solution, is indeed energetically and stereochemically favourable.

NMR and X-ray crystallographic studies on cyclic tetrapeptide, cyclo (D-Phe-Pro-Sar-Gly)

The conformation of the synthetic cyclic tetrapeptide cyclo(D-Phe-Pro-Sar-Gly) has been determined in solution using the nuclear magnetic resonance technique and in the crystal state by X-ray crystallography. Results showed that the peptide exhibited two different conformations in solution, conformer 1 having cis-trans-cis-trans peptide bonds and conformer 2 having trans-cis-trans-cis peptide bonds. No intramolecular hydrogen bonds were observed in the structures. The X-ray diffraction studies showed the crystals to be orthorhombic with space group P2(1)2(1)2(1) with unit-cell dimensions, a = 5.790, b = 10.344, c = 31.446 A, Z = 4, R = 0.104 for 2301 observed reflections. The crystal structure showed only one type of conformer having cis-trans-cis-trans peptide bonds similar to the conformer 1 in solution.

Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.

DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec: H1t is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

DNA-condensing and chromatin-condensing properties of rat testes hla and hit compared to those of rat-liver hlbdec - hlt is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

An incipient 310 helix in Piv-Pro-Pro-Ala-NHMe as a model for peptide folding

The molecular mechanism of helix nucleation in peptides and proteins is not yet understood and the question of whether sharp turns in the polypeptide backbone serve as nuclei for protein folding has evoked controversy1,2. A recent study of the conformation of a tetrapeptide containing the stereochemically constrained residue alpha-aminoisobutyric acid, both in solution and the solid state, yielded a structure consisting of two consecutive beta-turns, leading to an incipient 310 helical conformation3,4. This led us to speculate that specific tri- and tetra-peptide sequences may indeed provide a helical twist to the amino-terminal segment of helical regions in proteins and provide a nucleation site for further propagation. The transformation from a 310 helical structure to an alpha-helix should be facile and requires only small changes in the phi and psi conformational angles and a rearrangement of the hydrogen bonding pattern5. If such a mechanism is involved then it should be possible to isolate an incipient 310 helical conformation in a tripeptide amide or tetrapeptide sequence, based purely on the driving force derived from short-range interactions. We have synthesised and studied the model peptide pivaloyl-Pro-Pro-Ala-NHMe (compound I) and provide here spectroscopic evidence for a 310 helical conformation in compound I.

Rotational isomerism about the Ca-CO bond in proline derivatives. 1H and 13C NMR studies of benzyloxycarbonyl-Pro-N-methylamide and pivaloyl-Pro-N-methylamide

The 270 MHz 1H n.m.r. spectrum of benzyloxycarbonyl-Pro-N-methylamide in CDCl3 is exchange broadened at 293° K. Spectral lines due to two species are frozen out at 253° K and a dynamically averaged spectrum is obtained at 323° K. A selective broadening of the Cβ and Cγ resonances in the 13C n.m.r. spectrum is observed at 253° K, with a splitting of the Cβ and Cγ resonances into a pair of lines of unequal intensity. A similar broadening of Cβ and Cγ peaks is also detected in pivaloyl-Pro-N-methylamide where cis-trans interconversion about the imide bond is precluded by the bulky t-butyl group. The rate process is thus attributed to rotation about the Cα-CO bond (ψ) and a barrier (ΔG#) of 14kcal mol-1 is estimated. 13C n.m.r. data for pivaloyl-Pro-N-methylamide in a number of solvents is presented and the differences in the Cβ and Cγ chemical shifts are interpreted in terms of rotational isomerism about the Cα-CO bond.

X-Pro peptides: Synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)4methyl ester. Evidence for a novel helical structure

The synthesis of the octapeptide, benzyloxycarbonyl-(-aminoisobutyryl-L-prolyl)4-methyl ester [Z-(Aib-Pro)4-OMe] and an analysis of its solution conformation is reported. The octapeptide is shown to possess three strong intramolecular hydrogen bonds on the basis of studies of the solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. 13C studies are consistent with a structure involving only trans Aib-Pro bonds, while ir experiments support a hydrogen-bonded conformation. The Aib 3, 5, and 7 NH groups are shown to participate in hydrogen bonding. A 310 helical conformation compatible with the spectroscopic data is suggested. The proposed conformation consists of three type III -turns with Aib and Pro at the corners and stabilized by 4 1 intramolecular hydrogen bonds.

Monoclinic polymorph of Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe(anhydrous). Parallel packing of 3(10)-/alpha-helices and a transition of helix type

The structures of two crystal forms of Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe have been determined. The triclinic form (P1, Z = 1) from DMSO/H2O crystallizes as a dihydrate (Karle, Sukumar & Balaram (1986) Proc, Natl, Acad. Sci. USA 83, 9284-9288). The monoclinic form (P2(1), Z = 2) crystallized from dioxane is anhydrous. The conformation of the peptide is essentially the same in both crystal system, but small changes in conformational angles are associated with a shift of the helix from a predominantly alpha-type to a predominantly 3(10)-type. The r.m.s. deviation of 33 atoms in the backbone and C beta positions of residues 2-8 is only 0.29 A between molecules in the two polymorphs. In both space groups, the helical molecules pack in a parallel fashion, rather than antiparallel. The only intermolecular hydrogen bonding is head-to-tail between helices. There are no lateral hydrogen bonds. In the P2(1) cell, a = 9.422(2) A, b = 36.392(11) A, c = 10.548(2) A, beta = 111.31(2) degrees and V = 3369.3 A for 2 molecules of C60H97N11O13 per cell.