Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides.

The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094].

Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Saccharomyces cerevisiae tridecapeptide pheromone receptor.

Peptides representing both loop and the sixth transmembrane regions of the alpha-factor receptor of Saccharomyces cerevisiae were synthesized by solid-phase procedures and purified to near homogeneity. CD, nmr, and modeling analysis indicated that in aqueous media the first extracellular loop peptide E1(107-125), the third intracellular loop peptide I3(231-243), and the carboxyl terminus peptide I4(350-372) were mostly disordered. In contrast, the second extracellular loop peptide E2(191-206) assumed a well-defined structure in aqueous medium and the sixth transmembrane domain peptide receptor M6(252-269, C252A) was highly helical in trifluoroethanol/water (4:1), exhibiting a kink at Pro258. A synthetic peptide containing a sequence similar to that of the sixth transmembrane domain of a constitutively active alpha-factor receptor M6(252-269, C252A, P258L) in which Leu replaces Pro258 exhibited significantly different biophysical properties than the wild-type sequence. In particular, this peptide had very low solubility and gave CD resembling that of a beta-sheet structure in hexafluoroacetone/water (1:1) whereas the wild-type peptide was partially helical under identical conditions. These results would be consistent with the hypothesis that the constitutive activity of the mutant receptor is linked to a conformational change in the sixth transmembrane domain. The study of the receptor segments also indicate that peptides corresponding to loops of the alpha-factor receptor do not appear to assume turn structures.

PTR3, a novel gene mediating amino acid-inducible regulation of peptide transport in Saccharomyces cerevisiae.

We have isolated and characterized the Saccharomyces cerevisiae PTR3 gene by functional complementation of a mutant deficient for amino acid-inducible peptide transport. PTR3 is predicted to encode a protein of 678 amino acids that exhibits no similarity to any other protein in the database. Deletion of the PTR3 open reading frame pleiotropically reduced the sensitivity to toxic peptides and amino acid analogues. Initial rates of radiolabelled dipeptide uptake demonstrated that elimination of PTR3 resulted in the loss of amino acid-induced levels of peptide transport. PTR3 was required for amino acid-induced expression of PTR2, the gene encoding the dipeptide/tripeptide transport protein, but was not necessary for nitrogen catabolite repression of peptide import or PTR2 expression. It was determined that PTR3 also modulates expression of BAP2, the gene encoding the branched-amino acid permease. Furthermore, we present genetic evidence that suggests that PTR3 functions within a novel regulatory pathway that facilitates amino acid induction of the PTR system.

Schizosaccharomyces pombe isp4 encodes a transporter representing a novel family of oligopeptide transporters.

We have recently cloned an oligopeptide transport gene from Candida albicans denoted OPT1. This gene showed significant sequence similarity to three open reading frames (ORFs) with no previously established function: isp4 from Schizosaccharomyces pombe and Saccharomyces cerevisiae YJL212C and YPR194C, identified during the genome project. The S. pombe gene isp4 was originally identified by Sato et al. as a gene that was upregulated through nitrogen starvation induction of meiosis. However, an isp4delta strain exhibited a wild-type phenotype with respect to sexual differentiation. We have found that the same isp4delta strain is deficient in tetrapeptide transport activity as measured by its resistance to toxic tetrapeptides, by its inability to accumulate a radiolabelled tetrapeptide and by the inability to use tetrapeptides as a sole source of an amino acid to satisfy an auxotrophic requirement. Similarly, we found that the ORF YPR194C from S. cerevisiae encodes an oligopeptide transporter. Sequence analyses as well as physiological evidence has led us to propose that the proteins encoded by isp4 and the genes identified from S. cerevisiae and C. albicans comprise a new group of transporters specific for small oligopeptides, which we have named the OPT family.

Preparation of radiolabeled peptides via an iodine exchange reaction.

Peptides labeled with radioactive 125I can be detected at the extremely low concentrations necessary for receptor binding studies and medical applications. Traditional methods of iodination often lead to inactive peptides due to excessive iodination, nonspecific iodination, or oxidative damage to the peptide. In this work these disadvantages are circumvented by labeling 127I containing peptides of a predetermined biological activity using a radioactive 125I exchange reaction with Na125I. Specific radioactivity up to 9.8 Ci/mmol was reached in a simple and efficient procedure.

Synthesis of alpha-factor analogues containing photoactivatable and labeling groups.

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing both p-benzoyl phenylalanine (Bpa), a photoactivatable group, and 3-(mono- or di-iodo-4-hydroxyphenyl)propanoic acid (iodinated HPP) or biotin as a tag, were synthesized using solid-phase methodologies on a [phenylacetamido]-methyl (PAM) resin. Bpa was introduced into the peptides using Bpa-hydroxybenzotriazole active ester during peptide chain assembly. Biotinylated alpha-factor analogues were prepared by assembling the desired peptide on the resin, and then reacting a specific amino group either with the symmetrical anhydride of biotin or with biotin using BOP as the activating agent prior to anhydrous hydrogen fluoride cleavage. Iodinated HPP was incorporated by acylating free peptides with Bolton-Hunter reagent (3-[diiodo-4-hydroxyphenyl]propanoic acid hydroxysuccinimide ester) in N,N-dimethylformamide and borate buffer (pH 8.0) solutions. Purification of all peptides to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase mu-Bondapak C18 column with acetonitrile/water/trifluoroacetic acid as the mobile phase. All products were characterized by amino acid analysis and fast atom bombardment mass spectrometry. Two analogues, alpha-(diiodotyrosine)-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr-O H, and epsilon-(diiodo-HPP)-Lys-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr -OH, were one twentieth to one-fortieth as active as a alpha-factor, and exhibited approximately one order of magnitude lower affinity to the alpha-factor receptor. The results suggest that these two analogues are alpha-factor agonists and that they can be used as probes of the alpha-factor receptor.

Conformational analysis of the Saccharomyces cerevisiae tridecapeptide mating pheromone by 13C,15N rotational-echo double resonance nuclear magnetic resonance spectroscopy.

The solid-state conformation of [Nle12]alpha-factor, the Saccharomyces cerevisiae tridecapeptide mating pheromone (WHWLQLKPGQPNleY), was investigated by 13C,15N rotational-echo double resonance (REDOR) nuclear magnetic resonance spectroscopy (NMR). Previous high-resolution NMR studies of [Nle12]alpha-factor in solution revealed a transient Type II beta-turn spanning residues 7-10 of the peptide. To investigate this region of [Nle12]alpha-factor in the solid state, a series of four selectively 13C,15N-enriched tridecapeptides were synthesized by solid-phase methods. Carbon-nitrogen distances between the labeled sites in lyophilized samples of [Nle12]alpha-factor were accurately measured by REDOR NMR. Experimentally determined distances were compared with those from calculated models for Type I and Type II beta-turns and for an extended chain. The measured distances indicate that, in a lyophilized powder, the central region of the [Nle12]alpha-factor is not in an extended conformation. The experimental data was most consistent with distances obtained from a distorted Type I beta-turn model.