Fluorescence microscopy methods in the study of protein structure and function.
As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we describe two fluorescence microscopic methodologies applied to the outer hair cell-specific membrane protein prestin: the intensity and fluorescence lifetime (FLIM) variants of FRET (Fluorescence Resonance Energy Transfer), used in the study of protein-protein interactions, and the Scanning Cysteine Accessibility Method (SCAM), used for the determination of protein conformation. The methods are readily adaptable to other proteins.
Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, USA.
Heather
H
Jensen-Smith
Benjamin
B
Currall
Danielle
D
Rossino
LeAnn
L
Tiede
Michael
M
Nichols
Richard
R
Hallworth
D000818:Animals; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000502:physiology*; D018072:Hair Cells, Auditory, Outer / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation
D020409:Molecular Motor Proteins; C498720:Pres protein, mouse; D049452:Green Fluorescent Proteins