The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide–protein and peptide–nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future

Accommodation of a D-Phe Residue into a Right-Handed 310-Helix: Structure of Boc-D-Phe- ( Aib)4-Gly-L-Leu-(Aib)2-OMe, an Analogue of the Amino Terminal Segment of Antiamoebins and Emerimicins

The crystal structure of the nonapeptide Boc-D-Phe-Aib-Aib-Aib-Aib-Gly-Leu-Aib-Aib-OMe (I), which is an analogue of the N-terminal sequence of antiamoebins and emerimicins, establishes a completely 310-helical conformation with seven successive intramolecular 4 - 1 hydrogen bonds. The average phi, psi values for residues 1-8 are –59 deg and –32 deg, respectively. Crystal parameters are C47H77N9O12, space group P1, a = 10.636(4) Å, b = 11.239(4) Å, c = 12.227(6) Å, alpha = 101.17(4) deg Å , beta = 97.22(4) deg, gamma = 89.80(3) deg, Z = 1, R = 5.95% for 3018 data with [Fo] > 3a(F), resolution 0.93 Å. The use of the torsion angle k = C ( i - l ) N ( i ) Calpha( i)Cbeta( i) , where k = 68" for D-Phe and k = 164' for L-Leu, confirms the opposite configurations of these residues. The phi, psi values of –62 deg and –32 deg at D-Phe are unusual, since this region is characteristic of residues with L configurations. Peptide I possesses only two chiral residues of opposing configuration. The observed right-handed 310-helical structure suggests that helix sense has probably been determined by the stereochemical preferences of the Leu residue

Conformation of a 16-residue zervamicin IIA analog peptide containing three different structural features: 310-helix, alpha helix and ß-bend ribbon

Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib- Pro-Ala-Aib-Pro-Aib-Pro-Phe-OM(we here Boc is t-butoxycarbonyla nd Aib is a-aminoisobutyriac cid), a synthetica polar analog of the membrane-activefu ngal peptide antibioticz ervamtycinII A, crystallizesi n spaceg roupP 1 withZ =1 and cell parameters a = 9.086 ?0.002 A, b = 10.410 ?+ 0.002 A, c = 28.188 ? 0.004 A, a = 86.13 ? 0.01?, 13 = 87.90 ? 0.01?, and y = 89.27 ? 0.01?;o veralla greementf actorR = 7.3% for 7180 data (Fo > 3cr) and 0.91-A resolution. The peptide backbone makes a continuous spiral that begins as a 310-helix at the N-terminus, changes to an a-helix for two turns, and ends in a spiral of three fl-bends in a ribbon. Each of the fl-bends contains a proline residue at one of the corners. The torsion angles 4i range from -51? to -91? (average value -64o), and the torsion angles ai range from -1? to -46? (average value -31?). There are 10 intramolecularN H...OCh ydrogenb onds in the helix and two directh ead-to-taihl ydrogenb ondsb etween successive molecules. Two H20 and two CH30H solvent molecules fill additional space with appropriate hydrogen bonding in the head-to-tail region, and two additional H20 molecules form hydrogen bonds with carbonyl oxygens near the curve in the helix at Pro-10. Since there is only one peptide molecule per cell in space group P1, the molecules repeat only by translation, and consequently the helices pack parallel to each other

Effects of End Group and Aggregation on Helix Conformation: Crystal Structure of Ac−(Aib−Val−Ala−Leu)2−Aib−OMeAc-(Aib-Val-Ala-Leu)_2-Aib-OMe

The role of end groups in determining stereochemistry and packing in hydrophobic helical peptides has been investigated using an \alpha -aminosobutyric acid (Aib) containing model nonapeptide sequence. In contrast to the Boc-analogue, $Ac-(Aib-Val-Ala-Leu)_2-Aib-OMe$ crystallizes with two independent molecules in a triclinic cell. The cell parameters are: space group P1, a=10.100(2)A, b=15.194(4) A, c=19.948(5) A, \alpha =63.12(2), \beta =88.03(2), \gamma =88.61(2), Z=2, R=7.96% for 5140 data where |Fo|>3\sigma (F). The two independent molecules alternate in infinite columns formed by head-to-tail hydrogen bonding. The helices in the two independent molecules are quite similar to each other but one molecule is rotated $\approx 123^ \circ$ about its helix axis with respect to the other. All the helical columns pack parallel to each other in the crystal. Replacement of the bulky Boc group does not lead to any major changes in conformation. Packing characteristics are also similar to those observed for similar helical peptides

Solvated Helical Backbones: X-Ray Diffraction Study of Boc-Ala-Leu-Aib-Ala-Leu-Aib-Ome.H2OH_2O

A second example of insertion of a water molecule into the helical backbone of an apolar peptide is presented here and compared to a similar occurrence in a longer peptide with the same type of sequence of residues, i.e., $Boc-Aib-{(Ala-Leu-fib)}_3-Ome$. The backbone of the title compound assumes an approximate $3_{10}$-helical form with three 4 \rightarrow 1 hydrogen bonds. In the place of a fourth 4 \rightarrow 1 hydrogen bond, a water molecule is inserJed between 0(1) and N(4), and acts as a bridge by forming hydrogen bonds N(4) . . . W(1) (2.95 A) and W(l) . . . O(1) (2.81 A). The water molecule participates in a third hydrogen bond with a neighboring peptide molecule, W(1) . . . O(4) (2.91 A). The insertion of the water molecule causes the apolar peptide to mimic an amphiphilic helix. Crystals grown from ethyl acetate/petroleum ether (reported here)p from methanol/water solution are in space group ${P2}_12_12_1$ with a = 12.024(4) A, b = 15.714(6) A, c = 21.411(7) A, Z = 4 and $d_{calc}$ = 1.124 g/$cm^3$ for $C_{32}H_{58}N_6O_9$ . $H_2O$. The overall agreement factor R is 6.3% for 2707 reflections observed with intensities > 3 \sigma (F) and the resolution is 0.90 A

Cystine peptides Antiparallel β-sheet conformation of the cyclic biscystine peptide [Boc-Cys-Ala-Cys-NHCH3]2

The crystal structure analysis of the cyclic biscystine peptide [Boc-Cys1-Ala2-Cys3-NHCH3]2 with two disulfide bridges confirms the antiparallel ?-sheet conformation for the molecule as proposed for the conformation in solution. The molecule has exact twofold rotation symmetry. The 22-membered ring contains two transannular NH ? OC hydrogen bonds and two additional NH ? OC bonds are formed at both ends of the molecule between the terminal (CH3)3COCO and NHCH3 groups. The antiparallel peptide strands are distorted from a regularly pleated sheet, caused mainly by the L-Ala residue in which ?=� 155° and ?= 162°. In the disulfide bridge C? (1)-C? (1)-S(1)-(3')-C?(3')-C?(3'), S�S = 2.030 Å, angles C? SS = 107° and 105°, and the torsional angles are �49, �104, +99, �81, �61°, respectively. The biscystine peptide crystallizes in space group C2 with a = 14.555(2) Ã…, b = 10.854(2) Ã…, c = 16.512(2)Ã…, and ?= 101.34(1) with one-half formula unit of C30H52N8O10S4· 2(CH3)2SO per asymmetric unit. Least-squares refinement of 1375 reflections observed with |F| > 3?(F) yielded an R factor of 7.2%

Aqueous channels within apolar peptide aggregates. Solvated helix of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib- -Ala-Leu-Aib-OMe. H2O.CH3OH

Although the peptide Boc-Aibl-Ala2-Leu3- Aib4-Alas Leu'-Aib7-Ala8-Leu9-Aib'0-OMe [with a t-butoxycarbonyl(Boc) blocking group at the amino terminus, a methyl ester (OMe) at the carboxyl terminus, and four a-aminoisobutyric (Aib) residues] has a 3-fold repeat of residues, the helix formed by the peptide backbone is irregular. The carboxyl-terminal half assumes an at-helical form with torsion angles ) and r of approximately -60° and -45°, respectively, whereas the amino-terminal half is distorted by an insertion of a water molecule between the amide nitrogen of Ala5 [N(5)] and the carbonyl oxygen of Ala2 [0(2)]. The water molecule W(1) acts as a bridge by forming hydrogen bonds N(5).W(1) (2.93 A) and W(1)---0(2) (2.86 A). The distortion of the helix exposes the carbonyl oxygens of Aib' and Aib4 to the outside environment, with the consequence that the helix assumes an amphiphilic character despite having all apolar residues. Neighboring helices in the crystal run in antiparallel directions. On one side of a helix there are only hydrophobic contacts with efficient interdigitation of leucine side chains with those from the neighboring helix. On the other side of the helix there are hydrogen bonds between protruding carbonyl oxygens and four water molecules that separate two neighboring helices. Along the helix axis the helices bind head-to-tail with a direct hydrogen bond N(2)-0(9) (3.00 A). Crystals grown from methanol/water solution are in space group P2, with a = 15.778 ± 0.004 A, b = 11.228 ± 0.002 A, c = 18.415 ± 0.003 A, = 102.10 ± 0.02ur and two formula units per cell for C49HON1003 2H2OCH3OH. The overall agreement factorR is 7.5% for 3394 reflections observed with intensities >3a(F), and the resolution is 0.90 A

The role of crystallography in drug design

Structure and function are intimately related. Nowhere is this more important than the area of bioactive molecules. It has been shown that the enantioselectivity of an enzyme is directly related to its chirality. X-ray crystallography is the only method for determining the “absolute” configuration of a molecule and is the most comprehensive technique available to determine the structure of any molecule at atomic resolution. Results from crystallographic studies provide unambiguous, accurate, and reliable 3-dimensional structural parameters, which are prerequisites for rational drug design and structure-based functional studies

Zervamicins, a structurally characterised peptide model for membrane ion channels

Voltage dependent membrane channels are formed by the zervamicins, a group of α-aminoisobutyric acid containing peptides. The role of polar residues like Thr, Gln and Hyp in promoting helical bundle formation is established by dramatically reduced channel lifetimes for a synthetic apolar analog. Crystal structures of Leu1-zervamicin reveal association of bent helices. Polar contacts between convex faces result in an ‘hour glass’ like arrangement of an aqueous channel with a central constriction. The structure suggests that gating mechanisms may involve movement of the Gln11 carboxamide group. Gln3 may play a role in modulating the size of the channel mouth