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Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation.

ABSTRACT: p-Cyanophenylalanine is an extremely useful fluorescence probe of protein structure that can be recombinantly and chemically incorporated into proteins. The probe has been used to study protein folding, protein-membrane interactions, protein-peptide interactions, and amyloid formation; however, the factors that control its fluorescence are not fully understood. Hydrogen bonding to the cyano group is known to play a major role in modulating the fluorescence quantum yield, but the role of potential side-chain quenchers has not yet been elucidated. A systematic study of the effects of different side chains on p-cyanophenylalanine fluorescence is reported. Tyr is found to have the largest effect followed by deprotonated His, Met, Cys, protonated His, Asn, Arg, and protonated Lys. Deprotonated amino groups are much more effective fluorescence quenchers than protonated amino groups. Free neutral imidazole and hydroxide ion are also effective quenchers of p-cyanophenylalanine fluorescence with Stern-Volmer constants of 39.8 and 22.1 M(-1), respectively. The quenching of p-cyanophenylalanine fluorescence by specific side chains is exploited in developing specific, high-sensitivity, fluorescence probes of helix formation. The approach is demonstrated with Ala-based peptides that contain a p-cyanophenylalanine-His or a p-cyanophenylalanine-Tyr pair located at positions i and i + 4. The p-cyanophenylalanine-His pair is most useful when the His side chain is deprotonated and is, thus, complementary to the Trp-His pair which is most sensitive when the His side chain is protonated.

SUBMITTER: Taskent-Sezgin H

PROVIDER: S-EPMC3199967 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation.

Taskent-Sezgin Humeyra H Marek Peter P Thomas Rosanne R Goldberg Daniel D Chung Juah J Carrico Isaac I Raleigh Daniel P DP

Biochemistry 20100701 29

p-Cyanophenylalanine is an extremely useful fluorescence probe of protein structure that can be recombinantly and chemically incorporated into proteins. The probe has been used to study protein folding, protein-membrane interactions, protein-peptide interactions, and amyloid formation; however, the factors that control its fluorescence are not fully understood. Hydrogen bonding to the cyano group is known to play a major role in modulating the fluorescence quantum yield, but the role of potentia ...[more]

PMID: 20565125

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Project description:Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.

Dataset Information

Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation.

Publications

Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation.

Similar Datasets

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