The speed limit for protein folding measured by triplet-triplet energy transfer.

A direct measure of intramolecular chain diffusion is obtained by the determination of triplet-triplet energy-transfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds (N) separating donor and acceptor and show a maximum for the shortest peptides (N = 3) with a time constant (tau = 1/k) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.

Using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer for combined in-source decay/post-source decay experiments.

Mass spectrometric experiments with fragment ions have not yet been possible with a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer because intense signals of fragment ions were rarely observed during continuous extraction and the mass resolution of in-source formed fragment ions has been low. This paper describes a combination of MALDI in-source decay and post-source decay experiments on a MALDI-TOF mass spectrometer equipped with delayed extraction. Fragment ions initially formed in the ion source were selected by the precursor ion gate and investigated by post-source decay. The in-source formed fragment ions were sufficiently excited to undergo further metastable decay. The new method was applied to linear peptides, the cyclic peptide gramicidin S and a pentasaccharide, leading to unambiguous structural information.

Side-chain effects on peptidyl-prolyl cis/trans isomerisation.

Peptidyl-prolyl cis/trans isomerisation has been frequently found as a rate limiting step in the folding of proteins. In order to determine whether the nature of the amino acid preceding proline controls the probability of cis prolyl bonds in native proteins, systematic studies on the thermodynamics and kinetics of the prolyl isomerisation in the pentapeptide series Ac-Ala-Xaa-Pro-Ala-Lys-NH2 were performed. All proteinogenic amino acids were substituted in the position preceding proline. When measured by 1H-NMR and CD spectroscopy both isomers proved to be devoid of ordered structure in the whole series of the oligopeptides in aqueous solution. Thus, isomerization rates and cis/trans ratios calculated from solvent jump and 1H-NMR magnetisation transfer experiments exclusively reflect the side-chain effects of the Xaa position in the peptide series. There is a rough correlation between the cis content in the oligopeptides and the propensity of Xaa-Pro cis prolyl bonds in proteins. This correlation suggests that the prolyl bond conformation is mainly determined by local effects in proteins. The rate constants kc-->t of pentapeptides containing unionised amino acids preceding proline range from 3.2 x 10(-3) s-1 (Xaa = Ala) to 0.5 x 10(-3) s-1 (Xaa = Trp) at 4 degrees C. Proline clustering led to an isomerisation cycle indicating considerable influence on the isomerisation rates of the peptide bond conformations flanking the rotating bond. Both tyrosine and histidine specifically reduce isomerisation rates severalfold by deprotonation of their respective side-chains.

The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides.

Peptide-4-nitroanilides can be quickly synthesised using an Fmoc-based approach on 2-chlorotritylchloride resin. Preformed building blocks Fmoc-Xaa-NH-Np (Xaa = Cit, Cys, Gln, His, Lys, Orn, Ser, Thr, Tyr, Trp) can be attached via side chain to the 2-chlorotritylchloride linker of the resin. N-terminal elongation yields the respective peptide-4-nitroanilides after detachment from the solid support. We synthesised a set of tetrapeptide-4-nitroanilides with the general structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Asp, Cit, Cys, Glu, Gln, His, Lys, Orn, Ser, Thr, Tyr, Trp). Even peptidyl-arginine-4-nitroanilides are available by a slightly modified procedure. First, the appropriate ornithine-containing peptide was synthesised. After detachment of the peptide from the resin the side-chain primary amino group was transformed to the guanidino function of arginine using 1-guanyl-3,5-dimethylpyrazole. A further application of this method is the convenient synthesis of phosphorylated peptide-4-nitroanilides. Five phosphopeptides with the general structure Ac-Ala-Xaa(PO3H2)-Pro-Yaa-NH-Np (Xaa = Ser, Thr, Tyr; Yaa = Tyr, Lys) and their nonphosphorylated analogues were prepared. Global phosphorylation was carried out on the resin-bound peptides using dibenzyl-N, N-diisopropyl-phosphoramidite/tetrazole followed by oxidation with tert-butyl hydroperoxide.

Conformational state of a 25-mer peptide from the cyclophilin-binding loop of the HIV type 1 capsid protein.

Recently a 25-residue part of Gag polyprotein from HIV type 1 (HIV-1) was reported to bind to the cytosolic 18 kDa cyclophilin (Cyp18) with an IC50 value of 180 microM. This peptide corresponds to the Cyp18-binding domain of HIV-1 Gag. A replacement of Gly with Ala in the cyclophilin-binding loop of HIV-1 Gag polyprotein results in the prevention of the packaging of Cyp18 into virions. We found only two conformers of this peptide among 16 possible expected conformers, owing to cis/trans isomerization of four peptidyl-prolyl bonds. Although this finding implicates the existence of a stabilizing structure, we were not able to detect secondary structure formation by 1H-NMR and CD spectroscopy. We characterized the peptide as a substrate for Cyp18 by two-dimensional exchange 1H-NMR spectroscopy. Surprisingly, we found similar binding characteristics for a peptide corresponding to 25-mer peptide containing the above-mentioned Gly to Ala substitution.

Extended binding sites of cyclophilin as revealed by the interaction with HIV-1 Gag polyprotein derived oligopeptides.

Oligopeptides derived from the gag polyprotein (Pr55gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cyp18). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag218-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-1 Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The IC50 value of 184 microM for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1-4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPIase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cyp18, and may add a previously unrecognized topological component to the known subsite specificity of cyclophilins.

Influence of helix formation on cis/trans isomerism of Xaa-Pro bonds flanking the helical segment.

The trifluoroethanol (TFE)-induced formation of alpha-helical structures in the peptide hormone calcitonin (salmon) was studied using limited proteolysis combined with capillary zone electrophoresis. A low TFE content in TFE/buffer mixtures was insufficient to introduce secondary structure, but two Lys-Leu bonds were found to have become inaccessible to proteolysis with clostripain. The influence of increasing helical degree of the Thr6-Lys18 (or Thr6-Tyr22 in the trans conformation) segment on the cis/trans isomerization of the adjacent Tyr22-Pro23 peptide bond was examined by means of isomer specific proteolysis. Results indicate that the helix dipole does not influence the cis/trans equilibrium distribution of the flanking Tyr22-Pro23 bond but considerably increases its isomerization rate Ko-->t.