De novo design: backbone conformational constraints in nucleating helices and β-hairpins

A modular approach to synthetic protein design is being developed using conformationally constrained amino acid as stereochemical directors of polypeptide chain folding. An overview of studies aimed at constructing peptide helices using α,α-dialkyated residues and β-hairpins using D-Pro as a turn nucleator is presented. The construction of helix-helix motifs and three- and four-stranded structures has been achieved using non-protein amino acids to stabilize specific elements of secondary structures

Aggregation of apolar peptides in organic solvents. Concentration dependence of <SUP>1</SUP>H-nmr parameters for peptide NH groups in 3<SUB>10</SUB> helical decapeptide fragment of suzukacillin

Peptide NH chemical shifts and their temperature dependences have been monitored as a function of concentration for the decapeptide, Boc-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-OMe in CDCl3(0.001-0.06M) and (CD3)2SO (0.001-0.03M). The chemical shifts and temperature coefficients for all nine NH groups show no significant concentration dependence in (CD3)2SO. Seven NH groups yield low values of temperature coefficients over the entire range, while one yields an intermediate value. In CDCl3, the Aib(1) NH group shows a large concentration dependence of both chemical shift and temperature coefficient, in contrast to the other eight NH groups. The data suggest that in (CD3)2SO, the peptide adopts a 310 helical conformation and is monomeric over the entire concentration range. In CDCl3, the 310 helical peptide associates at a concentration of 0.01M, with the Aib(1) NH involved in an intermolecular hydrogen bond. Association does not disrupt the intramolecular hydrogen-bonding pattern in the decapeptide

Structures of unliganded and inhibitor complexes of W168F, a loop6 hinge mutant of Plasmodium falciparum Triosephosphate Isomerase: observation of an intermediate position of loop6

The enzymatic reaction of triosephosphate isomerase (TIM) is controlled by the movement of a loop (loop6, residues 166-176). Crystal structures of TIMs from a variety of sources have revealed that the loop6, which is in an open conformation in the unliganded enzyme, adopts a closed conformation in inhibitor complexes. In contrast, structures with loop open conformation are obtained in most of the complexes of TIM from the malarial parasite Plasmodium falciparum (PfTIM). W168 is a conserved N-terminal hinge residue, involved in different sets of interactions in the "open" and "closed" forms of loop6. The role of W168 in determining the loop conformation was examined by structural studies on the mutant W168F and its complexes with ligands. The three-dimensional structures of unliganded mutant (1.8 Å) and complexes with sulfate (2.8 Å) and glycerol-2-phosphate (G2P) (2.8 Å) have been determined. Loop6 was found disordered in these structures, reflecting the importance of W168 in stabilizing either the open or the closed states. Critical sequence differences between the Plasmodium enzyme and other TIMs may influence the equilibrium between the closed and open forms. Examination of the environment of the loop6 shows that its propensity for the open or the closed forms is influenced not only by Phe96 as suggested earlier, but also by Asn233, which occurs in the vicinity of the active site. This residue is Gly in the other TIM sequences and probably plays a crucial role in the mode of ligand binding, which in turn affects the loop opening/closing process in PfTIM

Solution phase synthesis of alamethicin I

The total synthesis of alamethicin I by solution phase methods is reported

Stereochemical control of peptide folding

Stereochemically constrained amino acid residues that strongly favour specific backbone conformations may be used to nucleate and stabilize specific secondary structures in designed peptides. An overview of the use of &#945;&#945;-dialkyl amino acids in stabilizing helical structures in synthetic peptides is presented, with an emphasis on work carried out in the authors laboratory. &#945;-Aminoisobutyric acid (Aib) and related achiral homologs facilitate stable helix formation in oligopeptides as exemplified by a large number of crystal structure determinations in the solid state. The ability to design conformationally rigid helical modules has been exploited in attempts to design structurally well characterized helix-linker-helix, using potential nonhelical linking segments. &#946;-Hairpin design has been approached by exploiting the tendency of 'prime turns' to nucleate hairpin formation. The use of nucleating DPro-Gly segments has resulted in the generation of several well characterized &#946;-hairpin structures, including the crystallographic observation of &#946;-hairpin in a synthetic apolar octapeptide. Extensions of this approach to three stranded &#946;-sheets and larger structures containing multiple DPro-Gly segments appear readily possible

Stabilization of β-turn conformations in Pro-X sequences by disulphide bridging. Synthesis and solution conformations of five cyclic cystine peptides

Five cyclic peptide disulphides of the type Image have been synthesized, where X = Gly (1), L-Ala (2), D-Ala (3), Aib (4) and L-Leu (5). 1H NMR studies at 270 MHz in CDCl3 and (CD3)2SO provide evidence of a Pro-X &#946;-turn conformation, stabilized by a transannular 4&#8594;1 hydrogen bond involving the Cys(4) NH, in all the peptides. In addition peptides 2, 4 and 5 also possess a second intramolecular hydrogen bond involving the -NHMe group. The spectroscopic data are consistent with a consecutive type III &#946;-turn conformation for peptides 2, 4 and 5, a type I(III) &#946;-turn structure for 1 and a type II turn for 3

Racemization at proline residues during peptide bond formation : a study of diastereomeric mixtures of synthetic alamethicin fragments by 270 MHz <SUP>1</SUP>H NMR

The stepwise synthesis of amino terminal pentapeptide of alamethicin, Z-Aib-Pro-Aib-Ala-Aib-OMe, by the dicyclohexylcarbodiimide mediated couplings leads to extensive racemization at the Ala and Pro residues. Racemization is largely suppressed by the use of additives like N-hydroxysuccinimide and 1-hydroxybenzotriazole. The presence of diastereomeric peptides may be detected by the observation of additional methyl ester and benzylic methylene signals in the 270 MHz 1H NMR spectra. Unambiguous spectral assignment of the signals to the diastereomers has been carried out by the synthesis and NMR studies of the D-Ala tetra and pentapeptides. The racemization at Pro is of particular relevance in view of the reported lack of inversion at C-terminal Pro on carboxyl activation

Interactions of the channel forming peptide alamethicin with artificial and natural membranes

Alamethicin and related α-aminoisobutyric acid peptides form transmembrane channels across lipid bilayers. This article briefly reviews studies on the effect of alamethicin on lipid phase transitions in lipid bilayers and on mitochondrial oxidative phosphorylation. Fluorescence polarization studies, employing 1,6-diphenyl-1,3,5-hexatriene as a probe, suggest that alamethicin fluidizes lipid bilayers below the phase transition t-emperature, but has little effect above the gel-liquid crystal transition point. Alamethicin is shown to function as an uncoupler of oxidative phosphorylation in rat liver mitochondria. The influence of alamethicin on mitochondrial respiration is modulated by the phosphate ion concentration in the medium. Classical uncoupling activity is evident at low phosphate levels while inhibitory effects set in at higher phosphate concentrations. Time-dependent changes in respiration rates following peptide addition are rationalized in terms of alamethicin interactions with mitochondrial membrane components

Conformational properties of hybrid peptides containing α- and ω-amino acids

This review briefly surveys the conformational properties of guest &#969;-amino acid residues when incorporated into host &#946;-peptide sequences. The results presented focus primarily on the use of &#946;- and &#947;-residues in &#945;&#969; sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between &#945;-peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and &#946;-hairpin conformations permits the characterization of backbone conformational parameters for &#946;- and &#947;-residues inserted into regular &#945;-polypeptide structures. Substituted &#946;- and &#947;-residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral &#946;,&#946;-disubstituted &#947;-amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C&#946;-C&#947; (&#952;1) and C&#945;-C&#946; (&#952;2) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures

X-Pro peptides: synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)<SUB>4</SUB>methyl ester. Evidence for a novel helical structure

The synthesis of the octapeptide, benzyloxycarbonyl-(&#945;-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 &#946;-turns with Aib and Pro at the corners and stabilized by 4&#8594; 1 intramolecular hydrogen bonds