John Cavanagh — Page 2

PubMed2002

Stoichiometries of protein-protein/DNA binding and conformational changes for the transition-state regulator AbrB measured by pseudo cell-size exclusion chromatography-mass spectrometry.

We have developed on-line pseudo cell-size exclusion chromatography-mass spectrometry (PsC-SEC-MS) for the rapid, real time analyses of noncovalently bound protein complexes. The methodology can be used to determine constituent components of such complexes, as well as exact stoichiometries. Furthermore, it enables the efficient determination of gross conformational changes upon complexation. The power of the new approach is demonstrated in the analysis of the global transition-state regulator AbrB and its complex with a target DNA sequence from the promoter sinIR. Using PsC-SEC-MS, we confirm that AbrB is assembled as a homotetramer and not as a homohexamer as previously suggested. Additionally, we show that AbrB binds to the sinIR DNA target element as a homotetramer, affording a 4:1 protein:DNA stoichiometry. Finally, we demonstrate that when the complex binds to sinIR, the hydrodynamic volume (size) of the complex is notably reduced compared to that of the apoprotein, indicating a protein conformational change.

Bacillus subtilis↗

PubMed2002

Acrylamide capture of DNA-bound complexes: electrophoretic purification of transcription factors.

We have developed a rapid nonradioactive electrophoretic technique to analyze proteins within DNA-binding complexes, acrylamide capture of DNA-binding complexes (ACDC), using Acrydite-linked DNA-binding targets. The method is highly sensitive and easily adaptable to virtually any protein-DNA interaction. The utility of this technique is illustrated using recombinant and full-length androgen receptors and associated co-regulatory proteins present within nuclear extracts. In brief proteins were incubated with DNA-binding targets in which one oligonucleotide was synthesized with an Acrydite moiety at the 5' end to allow for covalent linkage to acrylamide. Alternatively, gene promoter regions were amplified with an Acrydite-modified PCR primer to analyze protein-DNA complexes. The DNA-binding reaction was polymerized into an acrylamide matrix within the well of a precast gel. Proteins complexed to the Acrydite DNA are trapped and purified by the electrophoretic migration of unbound proteins. Proteins captured in the Acrydite-DNA can be eluted and identified by Western analysis or 2-D gel electrophoresis. The advantages of this technique are that it is rapid, adaptable, sensitive, unlimited by the size of the DNA or protein complex, and can be used to detect tertiary interactions with co-regulatory factors and unidentified proteins. These features make the ACDC technique a powerful tool for transcription factor research.

Acrylamide↗