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In proteins, proline-aromatic sequences exhibit increased frequencies of cis-proline amide bonds, via proposed C–H/π interactions between the aromatic ring and either the proline ring or the backbone C–Hα of the residue prior to proline. These interactions would be expected to result in tryptophan, as the most electron-rich aromatic residue, exhibiting the highest frequency of cis-proline. However, prior results from bioinformatics studies on proteins and experiments on proline-aromatic sequences in peptides have not revealed a clear correlation between the properties of the aromatic ring and the population of cis-proline. An investigation of the effects of aromatic residue (aromatic ring properties) on the conformation of proline-aromatic sequences was conducted using three distinct approaches: (1) NMR spectroscopy in model peptides of the sequence Ac-TGPAr-NH2 (Ar = encoded and unnatural aromatic amino acids); (2) bioinformatics analysis of structures in proline-aromatic sequences in the PDB; and (3) computational investigation using DFT and MP2 methods on models of proline-aromatic sequences and interactions. C–H/π and hydrophobic interactions were observed to stabilize local structures in both the trans-proline and cis-proline conformations, with both proline amide conformations exhibiting C–H/π interactions between the aromatic ring and Hα of the residue prior to proline (Hα-trans-Pro-aromatic and Hα-cis-Pro-aromatic interactions) and/or with the proline ring (trans-ProH-aromatic and cis-ProH-aromatic interactions). These C–H/π interactions were strongest with tryptophan (Trp) and weakest with cationic histidine (HisH+). Aromatic interactions with histidine were modulated in strength by His ionization state. Proline-aromatic sequences were associated with specific conformational poses, including type I and type VI β-turns. C–H/π interactions at the pre-proline Hα, which were stronger than interactions at Pro, stabilize normally less favorable conformations, including the ζ or αL conformations at the pre-proline residue, cis-proline, and/or the g+ χ1 rotamer or αL conformation at the aromatic residue. These results indicate that proline-aromatic sequences, especially Pro-Trp sequences, are loci to nucleate turns, helices, loops, and other local structures in proteins. These results also suggest that mutations that introduce proline-aromatic sequences, such as the R406W mutation that is associated with protein misfolding and aggregation in the microtubule-binding protein tau, might result in substantial induced structure, particularly in intrinsically disordered regions of proteins.
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Proline‐Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β‐Casein Containing Milks
Abstract The proline amino acid and prolyl residues of peptides/proteins confer unique biological and biochemical properties that motivates the development of proline‐selective analysis. The study focuses on one specific class of problem, the detection of single amino acid variants involving proline, and reports a Pro‐selective electrochemiluminescence (ECL) method. To develop this method, the A1‐/A2‐ variants of milk's β‐casein protein are investigated because it is a well‐established example and abundant samples are readily available. Specifically, β‐casein has 209 amino acids with 34 (or 35) proline residues: the A1‐variant has a Pro‐to‐His substitution at position 67 (relative to the A2 variant). The study shows that proline's strong luminescence allows the generic discrimination of: Pro from other amino acids; an A2‐oligopeptide from an A1‐oligopeptide; the A2‐β‐casein variant from the A1‐variant; and commercially‐available A2 milks from A1‐containing regular milks. The evidence indicates that luminescence depends on proline content and accessibility, as well as signal quenching. Compared to conventional immunoassays, the ECL method is simple, rapid, and inexpensive. Further, the ECL‐method is Pro‐selective (vs molecularly‐selective like typical immunoassays) which should make it broadly useful for studying the role of proline in biology and especially useful for tracking the digestion of proline‐rich proteins in the diet.
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- PAR ID:
- 10571188
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Science
- Volume:
- 12
- Issue:
- 5
- ISSN:
- 2198-3844
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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