Modulation of p-Cyanophenylalanine Fluorescence by Amino Acid Side Chains and Rational Design of Fluorescence Probes of α-Helix Formation

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• 作者：Humeyra Taskent-Sezgin ; Peter Marek ; Rosanne Thomas ; Daniel Goldberg ; Juah Chung ; Isaac Carrico ; Daniel P. Raleigh
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：July 27, 2010
• 年：2010
• 卷：49
• 期：29
• 页码：6290-6295
• 全文大小：877K
• 年卷期：v.49,no.29(July 27, 2010)
• ISSN：1520-4995

文摘

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.