Linked Life Data

9245428

Source:http://linkedlifedata.com/resource/pubmed/id/9245428

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
32
pubmed:dateCreated
1997-9-8
pubmed:abstractText
Tryptophan repressor (TR) from Escherichia coli is a homodimer whose highly helical subunits intertwine in a complex fashion. A monomeric version of Trp repressor has been constructed by introducing a pair of polar amino acids at the hydrophobic dimer interface. Analytical ultracentrifugation was used to show that the replacement of leucine at position 39 with glutamic acid results in a monomer/dimer equilibrium whose dissociation constant is 1.11 x 10(-)4 M at 25 degrees C and pH 7.6. Tryptophan fluorescence, both near- and far-UV circular dichroism, and NMR spectroscopies demonstrated that, at the micromolar concentrations where the monomer predominates, secondary and tertiary structure are present. Hydrophobic dye-binding experiments showed that nonpolar surface is accessible in the monomeric form. The urea-induced equilibrium unfolding of monomeric L39E TR was monitored by circular dichroism, fluorescence, and absorbance spectroscopies. Coincident transitions show that the urea denaturation process follows a simple two-state model involving monomeric native and unfolded forms. The free energy at standard state in the absence of denaturant was estimated to be 2.37 +/- 0.15 kcal mol-1, and the sensitivity of the unfolding transition to denaturant, the m value, was 0.86 +/- 0.04 kcal mol-1 M(urea)-1 at pH 7.6 and 25 degrees C. The thermal denaturation transition occurred over a broad temperature range, suggesting either that the enthalpy change is small or that intermediates may exist. Kinetic studies showed that both the refolding and unfolding of the monomer were complete in the mixing dead time of stopped-flow CD and fluorescence spectroscopy, 5 ms. These structural, thermodynamic, and kinetic results are very similar to those previously reported for an early, monomeric intermediate in the folding of the wild-type TR dimer [Mann, C. J., & Matthews, C. R. (1993) Biochemistry 32, 5282-5290]. The construction of a stable, monomeric form of TR that strongly resembles a transient folding intermediate should provide useful insights into the nature of the early events in the folding of TR.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
9941-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Construction and characterization of monomeric tryptophan repressor: a model for an early intermediate in the folding of a dimeric protein.
pubmed:affiliation
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.