Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Formation of truncated peptide by-products via sequence-specific formyl group transfer from Trp(For) residues to Nα in the course of Boc-SPPS.

(N(In))-Formyl protective group of tryptophan has been introduced as a base/nucleophile-labile protective group. It has long been known that a free Nα-amino group of the peptide can serve as a nucleophile: an irreversible formyl N(In) → NH(2) transfer is consistently observed when deformylation is performed last on an otherwise deprotected peptide that possesses free Nα-amino group. Obviously, this particular side reaction should be expected any time free amino group is exposed to Trp(For), but, at the best of our knowledge, has never been reported in the course of Boc-SPPS. In the present communication, we describe a set of appropriately designed model experiments that permitted to detect the title side reaction both in solution and in solid-phase reactions. We observed intermolecular formyl group transfer with a model compound, Trp(For)-NH(2). Importantly, we also observed this migration on solid support with the rate roughly estimated to be up to 1% of residues per minute. We also observed that the formyl-group transfer reaction occurred in a sequence-dependent manner and was suppressed to a non-detectable level using 'in situ neutralization' technique. Because this side reaction is sequence dependent, there might be situations when the rate of the formation of Nα -formyl termination by-products is significant. In other cases, the Nα -For truncated by-products would not contaminate the final peptide significantly but still could be a source of microheterogeneity.

High stability of the hinge region in the membrane-active peptide helix of zervamicin: paramagnetic relaxation enhancement studies.

Zervamicin IIB is a 16 amino acid peptaibol that forms voltage dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. Stability of the hinge region and intermolecular interactions were investigated in the N- and C-terminally spin-labelled peptide analogues. Intermolecular and intramolecular paramagnetic enhancement indicates that zervamicin behaves as a rigid helical rod in methanol solution. There are no high amplitude hinge-bending motions, and the peptaibol is monomeric up to concentration 1.5 mM. Stability of the hinge region illustrates the helix stabilising propensity of the Pro residue in membrane mimic environments and implies absence of significant conformational rearrangement due to voltage peptaibol activation.