Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references.

Sedimentation velocity (SV) is a method based on first principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures.

Planck-Benzinger thermal work function: thermodynamic characterization of the carboxy-terminus of p53 peptide fragments.

The thermodynamic parameters for six p53 carboxy-terminus peptide fragments as determined by analytical ultracentrifugal analysis were compared over the experimental temperature range of 275-310 K to evaluate the Gibbs free energy change as a function of temperature, ΔG°(T), from 0 to 400 K using our general linear third-order fitting function, ΔG°(T) = α + βT² + γT³. Data obtained at the typical experimental temperature range are not sufficient to accurately describe the variations observed in the oligomerization of these p53 fragments. It is necessary to determine a number of thermodynamic parameters, all of which can be precisely assessed using this general third-order linear fitting function.

Grb2 adaptor undergoes conformational change upon dimerization.

Grb2 is an adaptor protein that couples activated receptor tyrosine kinases to downstream effector molecules such as Ras and Akt. Despite being a central player in mitogenic signaling and a target for therapeutic intervention, the role of Grb2 oligomerization in cellular signaling is not well understood. Here, using the techniques of size-exclusion chromatography, mass spectrometry, analytical ultra-centrifugation and isothermal titration calorimetry, we demonstrate that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven without an unfavorable enthalpic change at physiological temperatures.

Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer.

The p53 tumor suppressor is a tetrameric transcriptional enhancer, and its activity is compromised by mutations that cause amino acid substitutions in its tetramerization domain. Here we analyze the biochemical and biophysical properties of peptides corresponding to amino acids 319-358 of wild-type human p53, which includes the tetramerization domain, and that of a cancer-derived mutant with valine substituted for glycine 334. Unlike the wild-type peptide, the G334V peptide forms amyloid fibrils by a two-step process under physiological conditions of temperature and pH.

Domain interactions within the Ski2/3/8 complex and between the Ski complex and Ski7p.

The Ski complex (composed of Ski3p, Ski8p, and the DEVH ATPase Ski2p) is a central component of the 3'-5' cytoplasmic mRNA degradation pathway in yeast. Although the proteins of the complex interact with each other as well as with Ski7p to mediate degradation by exosome, a 3'-exonuclease complex, the nature of these interactions is not well understood. Here we explore interactions within the Ski complex and between the Ski complex and Ski7p using a directed two-hybrid approach combined with coimmunoprecipitation experiments.

Gal repressor-operator-HU ternary complex: pathway of repressosome formation.

DNA transaction reactions require formation of nucleoprotein complexes that involve multifaceted DNA-protein and protein-protein interactions. Genetic and biochemical studies suggested that the higher order Gal repressosome structure, which governs the transcription of two tandem galpromoters in Escherichia coli, involves sequence-specific binding of GalR repressor dimers to two operators, O(E) and O(I), located 113 bp apart, binding of GalR to the sequence-nonspecific DNA binding protein HU, interaction of HU with an architecturally critical DNA site between the two operators, and interaction between two DNA-bound GalR dimers generating a loop of the intervening DNA segment. In this paper, we demonstrate and determine the thermodynamic parameters of several of these interactions, GalR dimer-O(E), GalR tetramerization, HU-GalR, and HU-GalR-O(E) interactions, by analytical ultracentrifugation, fluorescence anisotropy, and fluorescence resonance energy transfer.

Modern analytical ultracentrifugation in protein science: a tutorial review.

Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes.