Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals.
Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.
Weik
Martin
M
Laboratoire de Biophysique Moléculaire and Laboratoire de Cristallographie et Cristallogenèse des Protéines, UMR 5075, Institut de Biologie Structurale, 38027 Grenoble, France.
Vernede
Xavier
X
Royant
Antoine
A
Bourgeois
Dominique
D
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D002091:Butyrylcholinesterase / Q000737:chemistry; D018360:Crystallography, X-Ray; D019793:Fluorescein / Q000494:pharmacology; D005898:Glass / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014101:Torpedo / Q000378:metabolism; D014867:Water / Q000737:chemistry
D011506:Proteins; D014867:Water; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D009113:Muramidase; D005990:Glycerol; D019793:Fluorescein