Investigating structural changes induced by nucleotide binding to RecA using difference FTIR.
Nucleotide binding to RecA results in either the high-DNA affinity form (Adenosine 5'-triphosphate (ATP)-bound) or the more inactive protein conformation associated with a lower affinity for DNA (Adenosine 5'-diphosphate (ADP)-bound). Many of the key structural differences between the RecA-ATP and RecA-ADP bound forms have yet to be elucidated. We have used caged-nucleotides and difference FTIR in efforts to obtain a comprehensive understanding of the molecular changes induced by nucleotide binding to RecA. The photochemical release of nucleotides (ADP and ATP) from biologically inactive precursors was used to initiate nucleotide binding to RecA. Here we present ATP hydrolysis assays and fluorescence studies suggesting that the caged nucleotides do not interact with RecA before photochemical release. Furthermore, we now compare difference spectra obtained in H2O and D2O as our first attempt at identifying the origin of the vibrations influenced by nucleotide binding. The infrared data suggest that unique alpha-helical, beta structures, and side chain rearrangements are associated with the high- and low-DNA affinity forms of RecA. Difference spectra obtained over time isolate contributions arising from perturbations in the nucleotide phosphates and have provided further information about the protein structural changes involved in nucleotide binding and the allosteric regulation of RecA.
Butler
Blaine C
BC
Department of Chemistry, James Madison University, Harrisonburg, Virginia 22807, USA.
Hanchett
Ross H
RH
Rafailov
Helena
H
MacDonald
Gina
G
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006859:Hydrogen / Q000378:metabolism; D006868:Hydrolysis; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013997:Time Factors; D014867:Water / Q000737:chemistry / Q000378:metabolism
D009711:Nucleotides; D014867:Water; D000244:Adenosine Diphosphate; D006859:Hydrogen; D000255:Adenosine Triphosphate; D003903:Deuterium; D011938:Rec A Recombinases