Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
CONSTRUCTION AND CHARACTERIZATION OF MONOMERIC TRYPTOPHAN REPRESSOR -A MODEL FOR AN EARLY INTERMEDIATE IN THE FOLDING OF A DIMERIC PROTEIN
Autore:
SHAO X; HENSLEY P; MATTHEWS CR;
Indirizzi:
PENN STATE UNIV,DEPT CHEM UNIVERSITY PK PA 16802 PENN STATE UNIV,DEPT CHEM UNIVERSITY PK PA 16802 PENN STATE UNIV,CTR BIOMOL STRUCT & FUNCT UNIVERSITY PK PA 16802 SMITHKLINE BEECHAM PHARMACEUT,DEPT MACROMOL SCI KING OF PRUSSIA PA 19406
Titolo Testata:
Biochemistry
fascicolo: 32, volume: 36, anno: 1997,
pagine: 9941 - 9949
SICI:
0006-2960(1997)36:32<9941:CACOMT>2.0.ZU;2-G
Fonte:
ISI
Lingua:
ENG
Soggetto:
SIZE-EXCLUSION CHROMATOGRAPHY; MOLTEN GLOBULE STATE; TRP APOREPRESSOR; ESCHERICHIA-COLI; LAMBDA-REPRESSOR; STABILITY; COOPERATIVITY; EQUILIBRIUM; SUBUNIT; BINDING;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
54
Recensione:
Indirizzi per estratti:
Citazione:
X. Shao et al., "CONSTRUCTION AND CHARACTERIZATION OF MONOMERIC TRYPTOPHAN REPRESSOR -A MODEL FOR AN EARLY INTERMEDIATE IN THE FOLDING OF A DIMERIC PROTEIN", Biochemistry, 36(32), 1997, pp. 9941-9949

Abstract

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 modelinvolving 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) atpH 7.6 and 25 degrees C. The thermal denaturation transition occurredover a broad temperature range, suggesting either that the enthalpy change is small or that intermediates may exist. Kinetic studies showedthat both the refolding and unfolding of the monomer were complete inthe 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 earlyevents in the folding of TR.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/09/20 alle ore 11:59:12