We tested 42 tetrapeptides for their ability to bind to the rat brain p2l' protein farnesyltransferase as estimated by their ability to compete with p2lH"-in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-Al-A2-X, where positions Al and A2 are occupied by aliphatic amino acids and position X is-occupied by a COOHterminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the Al position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CARL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) protein y subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21K'-1B was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the protein farnesyltranferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids.A farnesyl residue is attached in thioether linkage to the COOH-terminal cysteine of a variety of intracellular membrane-associated proteins. The list includes cellular p21lS proteins (1), nuclear lamin B (2), and the 'y subunit of bovine transducin (3). This modification is also found on mating factors secreted by fungi (4,5). In each case the farnesylated cysteine is initially the fourth residue from the COOH terminus (for review, see ref. 6). Farnesylation is followed by proteolytic removal of the three terminal residues and carboxylmethylation of the cysteine. These reactions render the COOH termini hydrophobic, presumably facilitating the initial attachment of the proteins to cell membranes. In some instances the hydrophobicity is increased by palmitoylation of nearby cysteines. Inspection of the sequences of the known farnesylated proteins has defined a weak consensus sequence for farnesylation that consists of Cys-A1-A2-X, where positions Al and A2 are occupied by aliphatic amino acids and position X is occupied by an undefined amino acid (7, 8).The likely donor of farnesyl residues is farnesyl pyrophosphate (FPP), an intermediate in the synthesis of sterols and polyisoprenes in eukaryotic cells (9). Recently, we have isolated from rat brain an enzyme that transfers a farnesyl group from [3H]FPP to p2lHa-as protein (10,11). The purified enzyme preparation contains two proteins, each with an apparent molecular mass ofabout 50 kDa, that appear to form a heterodimer (10, 11). The enzyme recognizes sequences as short as four amino acids provided that cysteine is at the fourth position from the COOH terminus. Recognition was demonst...
Cyclic pentapeptides are excellent models for reverse turns and have been used extensively in our laboratory to explore the influence of different amino acid sequences on turn preference. This paper is divided into two parts: In the first, we review our previous studies of cyclic pentapeptides. We summarize work that demonstrates the range of conformations possible within the cyclic pentapeptide backbone, the importance of sequence chirality in determining the backbone fold, and the utility of these cyclic pentapeptides as models for various turns. In the second, we present new results on two cyclic pentapeptides that contain beta-turns with Pro-Ala or Pro-Asn sequences in the i + 1 and i + 2 positions. By stereochemical criteria, a type I beta-turn is expected to be preferred by such L-L sequences. On the other hand, in proteins Asn occurs frequently in the i + 2 position of type II turns. We asked whether the same propensity would be manifest in an isolated model peptide, and if so, what the interactions were that influenced the relative stability of the type I and type II turns. To address these questions we have compared the conformational behavior of two peptides: cyclo(Gly-Pro-Ala-D-Phe-Pro) and cyclo(D-Ala-Pro-Asn-Gly-Pro). From previous studies, we anticipated that both peptides would contain an inverse gamma-turn and a beta-turn which consisted of either Gly-Pro-Ala-D-Phe or D-Ala-Pro-Asn-Gly in positions i to i + 3, respectively. Nuclear magnetic resonance analysis confirms this overall backbone conformation. Furthermore, quantitative nuclear Overhauser effect measurements in combination with molecular dynamics simulations and torsionally-forced energy minimizations have enabled us to determine that both type I and type II beta-turns are present in equilibrium in these peptides. The introduction of Asn in position i + 2 shifts this equilibrium significantly towards type II. We have done preliminary assessment of the possible side-chain/backbone conformations that contribute to the shift in populations.
Protein farnesyltransferase catalyzes isoprenylation of the cysteine four residues from the C-terminus of several proteins including p21ras. Farnesylation is required for the transforming activity of Ras, and many efforts are underway to develop inhibitors of farnesyltransferase. We have used nuclear magnetic resonance spectroscopy to determine the farnesyltransferase-bound conformation of a heptapeptide substrate, KTKCVFM, which competes for the modification of p21Ha-ras in an in vitro assay. Analysis of transferred nuclear Overhauser effects reveals that the CVFM sequence of the peptide substrate is directly involved in binding to the enzyme and adopts a type I beta-turn conformation in the bound state. The present structural information should aid in the design of more effective inhibitors of the enzyme and in understanding the nature of the peptide binding site.
We have synthesized a peptide corresponding to the 25-residue signal sequence plus the first 28 residues ofthe Escherichia coli outer membrane protein LamB in order to explore the properties ofa signal sequence in the presence of the N-terminal region of its passenger. In the last few years
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