| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biophysical Journal 67: 2511-2521 (1994)
© 1994 the Biophysical Society
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232.
ABSTRACT
Multiphasic kinetics are often observed in stopped-flow investigations. To characterize further these kinetic phases, we have developed a methodology whereby fluorescence total intensity and anisotropy stopped-flow data can be combined in a single analysis. Fluorescence total intensity and anisotropy are highly interrelated and contain two very complementary forms of information. Total-intensity changes are useful in determining changes in populations with differing quantum yields, whereas anisotropy changes contain additional contributions caused by the rotational dynamics of the species. For cases in which the fluorescence quantum yield increases, the observed rate of anisotropy change will be more rapid than the total-intensity change, whereas in cases in which the total intensity decreases, the observed change in anisotropy will lag behind. In all cases, with quantum yield changes the stopped-flow anisotropy signals cannot be fit with models consisting of exponentials. Case studies examining these effects are described for the protein folding/refolding transitions of Staphylococcal nuclease and phosphoglycerate kinase. A multiphasic DNA exonuclease reaction using bacteriophage T4 DNA polymerase is also examined. In all of these cases, combined analysis of both data types revealed insights into reaction mechanism, which could not be obtained by either data type in isolation. Quantum yields and steady-state anisotropies associated with transiently populated intermediate species can be resolved. The data analysis methodologies described allow characterization of multiphasic reactions in terms of internally consistent kinetic rates, quantum yields, and steady-state anisotropies.
This article has been cited by other articles:
 |
|
 |
|
  S. Osvath, L. Herenyi, P. Zavodszky, J. Fidy, and G. Kohler Hierarchic Finite Level Energy Landscape Model: TO DESCRIBE THE REFOLDING KINETICS OF PHOSPHOGLYCERATE KINASE J. Biol. Chem., August 25, 2006; 281(34): 24375 - 24380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Osvath, G. Kohler, P. Zavodszky, and J. Fidy Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase Protein Sci., June 1, 2005; 14(6): 1609 - 1616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Y. Brewer, J. Malicka, P. J. Blackshear, and G. M. Wilson RNA Sequence Elements Required for High Affinity Binding by the Zinc Finger Domain of Tristetraprolin: CONFORMATIONAL CHANGES COUPLED TO THE BIPARTITE NATURE OF AU-RICH mRNA-DESTABILIZING MOTIFS J. Biol. Chem., July 2, 2004; 279(27): 27870 - 27877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Williams, A. K. Snyder, P. Kuzmic, M. O'Donnell, and L. B. Bloom Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp: I. TWO DISTINCT ACTIVITIES FOR INDIVIDUAL ATP SITES IN THE {gamma} COMPLEX J. Biol. Chem., February 6, 2004; 279(6): 4376 - 4385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Snyder, C. R. Williams, A. Johnson, M. O'Donnell, and L. B. Bloom Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp: II. UNCOUPLING THE {beta} AND DNA BINDING ACTIVITIES OF THE {gamma} COMPLEX J. Biol. Chem., February 6, 2004; 279(6): 4386 - 4393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Osvath and M. Gruebele Proline Can Have Opposite Effects on Fast and Slow Protein Folding Phases Biophys. J., August 1, 2003; 85(2): 1215 - 1222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Defais, E. Phez, and N. P. Johnson Kinetic Mechanism for the Formation of the Presynaptic Complex of the Bacterial Recombinase RecA J. Biol. Chem., January 31, 2003; 278(6): 3545 - 3551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pabst, M. Margittai, D. Vainius, R. Langen, R. Jahn, and D. Fasshauer Rapid and Selective Binding to the Synaptic SNARE Complex Suggests a Modulatory Role of Complexins in Neuroexocytosis J. Biol. Chem., March 1, 2002; 277(10): 7838 - 7848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Wilson, K. Sutphen, M. Moutafis, S. Sinha, and G. Brewer Structural Remodeling of an A + U-rich RNA Element by Cation or AUF1 Binding J. Biol. Chem., October 12, 2001; 276(42): 38400 - 38409. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. G. Reid, W. A. Fenton, A. L. Horwich, and E. U. Weber-Ban ClpA mediates directional translocation of substrate proteins into the ClpP protease PNAS, March 16, 2001; (2001) 71043698. [Abstract] [Full Text]
|
|
|
|
|
|
G. M. Wilson, Y. Sun, H. Lu, and G. Brewer Assembly of AUF1 Oligomers on U-rich RNA Targets by Sequential Dimer Association J. Biol. Chem., November 19, 1999; 274(47): 33374 - 33381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Bertram, L. B. Bloom, J. Turner, M. O'Donnell, J. M. Beechem, and M. F. Goodman Pre-steady State Analysis of the Assembly of Wild Type and Mutant Circular Clamps of Escherichia coli DNA Polymerase III onto DNA J. Biol. Chem., September 18, 1998; 273(38): 24564 - 24574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. B. Bloom, J. Turner, Z. Kelman, J. M. Beechem, M. O'Donnell, and M. F. Goodman Dynamics of Loading the beta Sliding Clamp of DNA Polymerase III onto DNA J. Biol. Chem., November 29, 1996; 271(48): 30699 - 30708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Tota, L. Xu, A. Sirotina, C. D. Strader, and M. P. Graziano Interaction of [fluorescein-Trp[IMAGE]]Glucagon with the Human Glucagon Receptor Expressed in Drosophila Schneider 2 Cells J. Biol. Chem., November 3, 1995; 270(44): 26466 - 26472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Wilson, K. Sutphen, K.-y. Chuang, and G. Brewer Folding of A+U-rich RNA Elements Modulates AUF1 Binding. POTENTIAL ROLES IN REGULATION OF mRNA TURNOVER J. Biol. Chem., March 16, 2001; 276(12): 8695 - 8704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. Wilson, K. Sutphen, S. Bolikal, K.-y. Chuang, and G. Brewer Thermodynamics and Kinetics of Hsp70 Association with A + U-rich mRNA-destabilizing Sequences J. Biol. Chem., November 21, 2001; 276(48): 44450 - 44456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. G. Reid, W. A. Fenton, A. L. Horwich, and E. U. Weber-Ban ClpA mediates directional translocation of substrate proteins into the ClpP protease PNAS, March 27, 2001; 98(7): 3768 - 3772. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
