Linked Life Data

8161526

Source:http://linkedlifedata.com/resource/pubmed/id/8161526

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
15
pubmed:dateCreated
1994-5-26
pubmed:abstractText
Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (epsilon ADP) and the ribose-modified fluorescent analogs 3'(2')-O-(N-methylantraniloyl)adenosine 5'-diphosphate (MANT-ADP), 3'-O-(N-methylantraniloyl)deoxyadenosine 5'-diphosphate (MANT-dADP), 3'-O-(N-methylantraniloyl)-deoxyadenosine 5'-triphosphate (MANT-dATP), and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP). The obtained data indicate contrasting differences between these two regions. Binding of epsilon ADP to the DnaB helicase causes only approximately 21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound epsilon ADP is characterized by a single lifetime of 24.2 +/- 0.6 ns, only slightly shorter than the fluorescent lifetime of the free epsilon ADP in solution (25.5 +/- 0.6 ns). Solute-quenching studies of bound epsilon ADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent. These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound epsilon ADP is 0.21 +/- 0.02, compared to the anisotropy of 0.3 of completely immobilized epsilon ADP at the same excitation wavelength (lambda ex = 325 nm, lambda em = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site. In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2' and/or 3' oxygens of the ribose, increases upon binding to DnaB by factors of approximately 4.7 (lambda ex = 408 nm) and approximately 2.6 (lambda ex = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by approximately 11 nm and that of MANT-ADP by approximately 12 nm. Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent.(ABSTRACT TRUNCATED AT 400 WORDS)
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/1,N(6)-ethenoadenosine diphosphate, http://linkedlifedata.com/resource/pubmed/chemical/2',3'-(O-(2,4,6-trinitrocyclohexadie..., http://linkedlifedata.com/resource/pubmed/chemical/3'-O-(N-methylanthraniloyl)adenosine..., http://linkedlifedata.com/resource/pubmed/chemical/Adenine, http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Diphosphate, http://linkedlifedata.com/resource/pubmed/chemical/Anthranilic Acids, http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins, http://linkedlifedata.com/resource/pubmed/chemical/DNA Helicases, http://linkedlifedata.com/resource/pubmed/chemical/DnaB Helicases, http://linkedlifedata.com/resource/pubmed/chemical/Fluorescent Dyes, http://linkedlifedata.com/resource/pubmed/chemical/Nucleotides, http://linkedlifedata.com/resource/pubmed/chemical/Ribose
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
19
pubmed:volume
33
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
4682-94
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
Structural characteristics of the nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein. Studies with ribose and base-modified fluorescent nucleotide analogs.
pubmed:affiliation
Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch at Galveston 77555-0653.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.