title: Efficient UV detection of protein crystals enabled by fluorescence excitation at wavelengths longer than 300 nm.
abstract: 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.
authors.affiliation: Institute of Biochemistry and Molecular Biology, University of Hamburg, c/o DESY, Notkestrasse 85, Building 22a, 22603 Hamburg, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Dierks
authors.forename: Arne
authors.initials: A
authors.lastname: Meyer
authors.forename: Dominik
authors.initials: D
authors.lastname: Oberthür
authors.forename: Gert
authors.initials: G
authors.lastname: Rapp
authors.forename: Howard
authors.initials: H
authors.lastname: Einspahr
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
mesh_terms: D000818:Animals; D003460:Crystallization; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D014466:Ultraviolet Rays
chemical_list: D011506:Proteins
references.citation: Anal Chem. 1995 Aug 1;67(15):2575-9
references.pmid: 8849024
references.citation: Curr Opin Struct Biol. 2000 Oct;10(5):558-63
references.pmid: 11042454
references.citation: Acta Crystallogr D Biol Crystallogr. 2007 Apr;63(Pt 4):526-35
references.pmid: 17372358
references.citation: Biochem J. 1981 May 1;195(2):481-4
references.pmid: 7316963
references.citation: J Struct Biol. 2003 Apr;142(1):154-61
references.pmid: 12718927
references.citation: Acta Crystallogr D Biol Crystallogr. 2006 Feb;62(Pt 2):146-50
references.pmid: 16421445
references.citation: Acta Crystallogr D Biol Crystallogr. 2005 Jan;61(Pt 1):60-6
references.pmid: 15608376
references.citation: Acta Crystallogr D Biol Crystallogr. 2002 Jun;58(Pt 6 Pt 2):921-7
references.pmid: 12037293
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Mar 1;66(Pt 3):364-72
references.pmid: 20208182
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Apr;60(Pt 4):696-702
references.pmid: 15039558
references.citation: Pharm Res. 2008 Jul;25(7):1487-99
references.pmid: 18172579
references.citation: Nat Rev Drug Discov. 2002 Jan;1(1):45-54
references.pmid: 12119609
references.citation: Appl Environ Microbiol. 2004 Jun;70(6):3650-63
references.pmid: 15184170