| | |  | | | | Record from Web of Science® | | | | | | |
| Construction and characterization of monomeric tryptophan repressor: A model for an early intermediate in the folding of a dimeric protein |
|
|
| Author(s): Shao X, Hensley P, Matthews CR |
| Source: BIOCHEMISTRY Volume: 36 Issue: 32 Pages: 9941-9949 Published: AUG 12 1997 |
| Times Cited: 31 References: 54 |
| 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 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. |
| Document Type: Article |
| Language: English |
Addresses:
1. PENN STATE UNIV, DEPT CHEM, UNIVERSITY PK, PA 16802 USA
2. PENN STATE UNIV, CTR BIOMOL STRUCT & FUNCT, UNIVERSITY PK, PA 16802 USA
3. SMITHKLINE BEECHAM PHARMACEUT, DEPT MACROMOL SCI, KING OF PRUSSIA, PA 19406 USA |
| Publisher: AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: XQ760 |
| ISSN: 0006-2960 |
|
| | | | | | | | | Record from Web of Science® | | | | | | | | | |