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- Thermal and Sodium Dodecylsulfate Induced Transitions of Str...Thermal and Sodium Dodecylsulfate Induced Transitions of Streptavidin
Biophysical Journal, Volume 87, Issue 4, 1 October 2004, Pages 2701-2713
Mark J. Waner, Irina Navrotskaya, Amanda Bain, Edward Davis Oldham and David P. Mascotti
AbstractThe strong specific binding of streptavidin (SA) to biotin is utilized in numerous biotechnological applications. The SA tetramer is also known to exhibit significant stability, even in the presence of sodium dodecylsulfate (SDS). Despite its importance, relatively little is known about the nature of the thermal denaturation pathway for SA. This work uses a homogeneous SA preparation to expand on the data of previous literature reports, leading to the proposal of a model for temperature induced structural changes in SA. Temperature dependent data were obtained by SDS and native polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorescence and ultraviolet (UV)-visible spectroscopy in the presence and absence of SDS. In addition to the development of this model, it is found that the major thermal transition of SA in 1% SDS is reversible. Finally, although SA exhibits significant precipitation at elevated temperatures in aqueous solution, inclusion of SDS acts to prevent SA aggregation.
Abstract | Full Text | PDF (495 kb) - Molecular Competition for NKG2DMolecular Competition for NKG2D
Immunity, Volume 15, Issue 2, 1 August 2001, Pages 201-211
Christopher A O'Callaghan, Adelheid Cerwenka, Benjamin E Willcox, Lewis L Lanier and Pamela J Bjorkman
SummaryNKG2D is a potent activating receptor on natural killer cells, T cells, and macrophages. Mouse NKG2D interacts with two cell surface ligands related to class I MHC molecules: RAE1 and H60. We used soluble versions of NKG2D, RAE1, and H60 to characterize their interactions. RAE1 and H60 each bind NKG2D with nanomolar affinities, indicating tighter binding than most cell surface immune interactions, but NKG2D binds to H60 with ∼25-fold higher affinity than to RAE1. RAE1 and H60 compete directly for occupancy of NKG2D, and, thus, NKG2D can be occupied by only one ligand at a time. The NKG2D-H60 interaction is more temperature dependent and makes greater use of electrostatic interactions than the NKG2D-RAE1 interaction. The distinct thermodynamic profiles provide insights into the different molecular mechanisms of the binding interactions.
Summary | Full Text | PDF (359 kb) - Binding Properties of HABA-Type Azo Derivatives to Avidin an...Binding Properties of HABA-Type Azo Derivatives to Avidin and Avidin-Related Protein 4
Chemistry & Biology, Volume 13, Issue 10, 1 October 2006, Pages 1029-1039
Susanna Repo, Tiina A. Paldanius, Vesa P. Hytönen, Thomas K.M. Nyholm, Katrin K. Halling, Juhani Huuskonen, Olli T. Pentikäinen, Kari Rissanen, J. Peter Slotte, Tomi T. Airenne, Tiina A. Salminen, Markku S. Kulomaa and Mark S. Johnson
SummaryThe chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to -biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands β3 and β4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 Å) X-ray structure for avidin reported here. These results may be valuable in designing improved tools for avidin-based life science and nanobiotechnology applications.
Summary | Full Text | PDF (544 kb) - …more
Copyright © 1995 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 69, Issue 5, 2125-2130, 1 November 1995
doi:10.1016/S0006-3495(95)80083-4
Research Article
The relationship between ligand-binding thermodynamics and protein-ligand interaction forces measured by atomic force microscopy
A. Chilkoti, T. Boland, B.D. Ratner and P.S. Stayton
Center for Bioengineering, University of Washington, Seattle 98195, USA.
Abstract
The interaction forces between biotin and a set of streptavidin site-directed mutants with altered biotin-binding equilibrium and activation thermodynamics have been measured by atomic force microscopy. The AFM technique readily discriminates differences in interaction force between the site-directed (Trp to Phe or Ala) mutants. The interaction force is poorly correlated with both the equilibrium free energy of biotin binding and the activation free energy barrier to dissociation of the biotin-streptavidin complex. The interaction force is generally well correlated with the equilibrium biotin-binding enthalpy as well as the enthalpic activation barrier, but in the one mutant where these two parameters are altered in opposite directions, the interaction force is clearly correlated with the activation enthalpy of dissociation. These results suggest that the AFM force measurements directly probe the enthalpic activation barrier to ligand dissociation.
