Probing protein electrostatics with a synthetic fluorescent amino acid.
Electrostatics affect virtually all aspects of protein structure and activity and are particularly important in proteins whose primary function is to stabilize charge. Here we introduce a fluorescent amino acid, Aladan, which can probe the electrostatic character of a protein at multiple sites. Aladan is exceptionally sensitive to the polarity of its surroundings and can be incorporated site-selectively at buried and exposed sites, in both soluble and membrane proteins. Steady-state and time-resolved fluorescence measurements of Aladan residues at different buried and exposed sites in the B1 domain of protein G suggest that its interior is polar and heterogeneous.
Cohen
Bruce E
BE
Howard Hughes Medical Institute and Departments of Physiology and Biochemistry, University of California San Francisco, San Francisco, CA 94143, USA. bcohen@itsa.ucsf.edu
McAnaney
Tim B
TB
Park
Eun Sun
ES
Jan
Yuh Nung
YN
Boxer
Steven G
SG
Jan
Lily Yeh
LY
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*; D000409:Alanine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000138:chemical synthesis / Q000737:chemistry*; D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry*; D004121:Dimethyl Sulfoxide; D005453:Fluorescence; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D015221:Potassium Channels / Q000737:chemistry / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000502:physiology; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D051637:Shaker Superfamily of Potassium Channels; D012997:Solvents; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D000596:Amino Acids; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins; D051637:Shaker Superfamily of Potassium Channels; D012997:Solvents; C459091:aladan; D015081:2-Naphthylamine; D000409:Alanine; D004121:Dimethyl Sulfoxide