Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study.
The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.
Taskent-Sezgin
Humeyra
H
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
Chung
Juah
J
Patsalo
Vadim
V
Miyake-Stoner
Shigeki J
SJ
Miller
Andrew M
AM
Brewer
Scott H
SH
Mehl
Ryan A
RA
Green
David F
DF
Raleigh
Daniel P
DP
Carrico
Isaac
I
D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009570:Nitriles / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics; D012997:Solvents; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D010649:Phenylalanine / Q000031:analogs & derivatives
D000409:Alanine; D009570:Nitriles; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D012997:Solvents; C530634:p-cyanophenylalanine; D010649:Phenylalanine; C049650:ribosomal protein L9