Efficient UV detection of protein crystals enabled by fluorescence excitation at wavelengths longer than 300 nm.
It is well known that most proteins and many other biomolecules fluoresce when illuminated with UV radiation, but it is also commonly accepted that utilizing this property to detect protein crystals in crystallization setups is limited by the opacity of the materials used to contain and seal them. For proteins, this fluorescence property arises primarily from the presence of tryptophan residues in the sequence. Studies of protein crystallization results in a variety of setup configurations show that the opacity of the containment hardware can be overcome at longer excitation wavelengths, where typical hardware materials are more transparent in the UV, by the use of a powerful UV-light source that is effective in excitation even though not at the maximum of the excitation response. The results show that under these circumstances UV evaluation of crystallization trials and detection of biomolecular crystals in them is not limited by the hardware used. It is similarly true that a deficiency in tryptophan or another fluorescent component that limits the use of UV light for these purposes can be effectively overcome by the addition of fluorescent prostheses that bind to the biomolecule under study. The measurements for these studies were made with a device consisting of a potent UV-light source and a detection system specially adapted (i) to be tunable via a motorized and software-controlled absorption-filter system and (ii) to convey the excitation light to the droplet or capillary hosting the crystallization experiment by quartz-fibre light guides.
Dierks
Karsten
K
Institute of Biochemistry and Molecular Biology, University of Hamburg, c/o DESY, Notkestrasse 85, Building 22a, 22603 Hamburg, Germany.
Meyer
Arne
A
Oberthür
Dominik
D
Rapp
Gert
G
Einspahr
Howard
H
Betzel
Christian
C
D000818:Animals; D003460:Crystallization; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D014466:Ultraviolet Rays
D011506:Proteins
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