1. S' subsite mapping of serine proteases based on fluorescence resonance energy transfer
S Grahn, T Kurth, H D Jakubke, D Ullmann Biochim Biophys Acta . 1999 May 18;1431(2):329-37. doi: 10.1016/s0167-4838(99)00059-x.
A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.
2. Synthesis and properties of an all-D model ribonuclease S-peptide
M A Corigliano-Murphy, C Ponnamperuma, I M Chaiken, T Kanmera, D Dalzoppo, A Fontana, L A Xun Int J Pept Protein Res . 1985 Mar;25(3):225-31. doi: 10.1111/j.1399-3011.1985.tb02168.x.
In order to examine the effect of a defined enantiomeric sequence on protein structure, the all-D model ribonuclease S-peptide, H-Ala-Glu-Ala4-Lys-Phe-Ala-Arg-Ala-His-Met-Ala2-OH, has been synthesized by the solid phase method. The all-L peptide has been synthesized previously and shown to possess 36% of ribonuclease S activity when added to ribonuclease S-protein (Komoriya, A. & Chaiken, I.M. (1982) J. Biol. Chem 257, 2599-2604). The synthetic D-peptide was purified by gel filtration and semipreparative reverse phase HPLC. Amino acid composition of the synthetic peptide was in agreement with theory and gas chromatographic analysis showed that no significant racemization had occurred during synthesis. Circular dichroism (CD) studies of the D-peptide showed a peak of positive ellipticity in the 220-230 nm region, whereas a negative ellipticity peak for the L-peptide was observed. The effects of temperature and trifluoroethanol on the far-ultraviolet CD spectra of D- and L-peptides were similar but of opposite sign, confirming the expectation that the D-peptide has the propensity to form an alpha-helical structure which is enantiomeric with respect to that formed by the L-peptide. In the presence of S-protein, the L-peptide showed hydrolytic activity against the substrate cytidine-2':3'-monophosphate, whereas the D-peptide was inactive. Addition of the D-peptide to mixtures of L-peptide and S-protein did not lead to inhibition of enzymatic activity. These results indicate lack of binding of D-peptide to S-protein to produce either an active or inactive species.
3. Covalent attachment of peptides to cytochrome C for automated sequence determination
R L Sloan, M D Garrick Prep Biochem . 1977;7(2):111-28. doi: 10.1080/00327487708061630.
Because small peptides are lost into the organic solvents used, it is virtually impossible to obtain the complete amino acid sequence of a small peptide using only an automated peptide sequencer of the spinning cup type. To overcome this problem we have extended peptides at the carboxy terminus by attachment to equine cytochrome c by a water soluble carbodiimide, relying on the acetylated N-terminus of the cytochrome to minimize its direct contribution to recovery of PTH-amino acids. The Model Peptide H-Leu-Trp-Met-Arg-phe-Ala-OH was used for most experiments. After reaction of 3H-peptide with cytochrome c, about one-third of the tritium counts migrated with cytochrome c during gel filtration. After attachment, the amino acid sequence of the hexapeptide was readily determined with a single cleavage Quadrol program in a Beckman 890B sequencer, whereas only the N-terminal residue was recovered without attachment. The repetitive yield after attachment was 95-96%, with 21-27+ overlap and an initial yield of 18-20%. Sequence data with other peptides illustrate applications and present limitations of our approach.
