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Record 3 of 2602
Fluorescence Studies of Protein Crystallization Interactions
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Author and Affiliation:
Pusey, Marc L.(NASA Marshall Space Flight Center, Huntsville, AL United States)
Smith, Lori(Universities Space Research Association, Huntsville, AL United States)
Forsythe, Elizabeth(Universities Space Research Association, Huntsville, AL United States)
Abstract: We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.
Publication Date: Jan 01, 1999
Document ID:
(Acquired Nov 05, 1999)
Document Type: Conference Paper
Meeting Information: 24 May 1999; Buffalo, NY; United States
Meeting Sponsor: American Crystallographic Association; Buffalo, NY United States
Financial Sponsor: NASA Marshall Space Flight Center; Huntsville, AL United States
Organization Source: NASA Marshall Space Flight Center; Huntsville, AL United States
Description: 1p; In English
Distribution Limits: Unclassified; Publicly available; Unlimited
Rights: No Copyright
Availability Notes: Abstract Only;
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