Linked Life Data

10521278

Source:http://linkedlifedata.com/resource/pubmed/id/10521278

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
41
pubmed:dateCreated
1999-11-17
pubmed:abstractText
Side chain mobility, accessibility, and backbone motion were studied by site-directed spin labeling of sequential cysteine mutants of the G strand in tear lipocalins (TL). A nitroxide scan between residues 98 and 105 revealed the alternating periodicity of mobility and accessibility to NiEDDA and oxygen, characteristic of a beta-strand. Residue 99 was the most inaccessible to NiEDDA and oxygen. EPR spectra with the fast relaxing agent, K(3)Fe(CN)(6), exhibited two nitroxide populations for most residues. The motionally constrained population was relatively less accessible to K(3)Fe(CN)(6) because of dynamic tertiary contact, probably with side chain residues of adjacent strands. With increasing concentrations of sucrose, the spectral contribution of the immobile component was greater, indicating a larger population with tertiary contact. Increased concentrations of sucrose also resulted in a restriction of mobility of spin-labeled fatty acids which were bound within the TL cavity. The data suggest that sucrose enhanced ligand affinity by slowing the backbone motion of the lipocalin. The correlation time of an MTSL derivative (I) attached to F99C resulted in the lack of side chain motion and therefore reflects the overall rotation of the TL complex. The correlation time of F99C in tears (13.5 ns) was the same as that in buffer and indicates that TL exists as a dimer under native conditions. TL-spin-labeled ligand complexes have a shorter correlation time than the protein alone, indicating that the fatty acids are not rigidly anchored in the cavity, but move within the pocket. This segmental motion of the ligand was modulated by protein backbone fluctuations. Accessibility studies with oxygen and NiEDDA were performed to determine the orientation and depth of a series of fatty acid derivatives in the cavity of TL. Fatty acids are oriented with the hydrocarbon tail buried in the cavity and the carboxyl group oriented toward the mouth. In general, the mobility of the nitroxide varied according to position such that nitroxides near the mouth had greater mobility than those located deep in the cavity. Nitroxides positioned up to 16 carbon units from the hydrocarbon tail of the ligand are motionally restricted and inaccessible, indicating the cavity extends to at least this depth. EPR spectra obtained with and without sucrose showed that the intracavitary position of lauric acid in TL is similar to that in beta-lactoglobulin. However, unlike beta-lactoglobulin, TL binds 16-doxyl stearic acid, suggesting less steric hindrance and greater promiscuity for TL.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
13707-16
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed-meshheading:10521278-Amino Acid Sequence, pubmed-meshheading:10521278-Binding Sites, pubmed-meshheading:10521278-Carrier Proteins, pubmed-meshheading:10521278-Circular Dichroism, pubmed-meshheading:10521278-Dimerization, pubmed-meshheading:10521278-Edetic Acid, pubmed-meshheading:10521278-Electron Spin Resonance Spectroscopy, pubmed-meshheading:10521278-Humans, pubmed-meshheading:10521278-Ligands, pubmed-meshheading:10521278-Lipocalin 1, pubmed-meshheading:10521278-Mutagenesis, Site-Directed, pubmed-meshheading:10521278-Oxygen, pubmed-meshheading:10521278-Peptide Fragments, pubmed-meshheading:10521278-Protein Structure, Secondary, pubmed-meshheading:10521278-Protein Structure, Tertiary, pubmed-meshheading:10521278-Recombinant Proteins, pubmed-meshheading:10521278-Spin Labels, pubmed-meshheading:10521278-Sucrose, pubmed-meshheading:10521278-Tears
pubmed:year
1999
pubmed:articleTitle
Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling.
pubmed:affiliation
Department of Pathology, University of California at Los Angeles School of Medicine 90095, USA. bglasgow@pathology.medsch.ucla.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't