Difference FT-IR Studies of Nucleotide Binding to the Recombination Protein RecA

Brewer, Scott H.; Cresawn, Steven G.; Nguyen, Duyen Thi Cam; MacDonald, Gina

doi:10.1021/jp001069w

Cited by 2 publications

(25 citation statements)

References 33 publications

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“…Under conditions previously used in our laboratory we have generated similar spectra to those presented in Fig. 3 (Brewer et al, 2000). However, in our previous data the photolysis of the caged-nucleotides resulted in a strong positive vibration around 1690 cm Ϫ1 that arises from the formation of a C ϭ O on the free cage (Brewer et al, 2000;Barth et al, 1991;Cepus et al, 1998b).…”

Section: Infrared Difference Spectrasupporting

confidence: 66%

“…Some additional assays were performed in the absence of DNA (37°C) with the same concentration of phosphenol pyruvate, NADH, pyruvate kinase, and lactate dehydrogenase. However, these assays were performed in either 100ϫ Hepes buffer M or 100ϫ buffer I that was used in previous infrared experiments (Brewer et al, 2000).…”

Section: Atp Hydrolysis Assaysmentioning

confidence: 99%

“…The Hepes buffer M was used for all infrared experiments presented here. Each infrared sample was prepared as described previously except for the addition of DTT to a final concentration of 10 mM in the infrared samples (Brewer et al, 2000). Infrared spectra were recorded using a 560 Magna spectrometer (Nicolet, Madison, WI) that was equipped with a Mercury cadmium telluride/A detector, cooled by liquid nitrogen.…”

Section: Infrared Studiesmentioning

confidence: 99%

“…Recent experiments in our laboratory suggest that our infrared samples contained ϳ100 times the original concentration of buffer and salts after dehydration. To discern whether previous conditions using Tris buffer (Brewer et al, 2000) or current conditions that use Hepes buffer could result in ATP hydrolysis in the absence of DNA, activity assays were performed in 100 times the concentration of the two infrared buffers. The activity assays revealed that in the presence of 100ϫ Tris buffer I (no DNA) only negligible ATPase activity is obtained when compared with the standard reaction conditions in the presence of polydT.…”

Section: Atp Hydrolysis and Fluorescence Datamentioning

confidence: 99%

“…The recent assays and previous studies performed by Ellouze et al (1995) imply that the protein may adopt a more extended conformation (especially after nucleotide binding) than present in our previous data. (Brewer et al, 2000) Furthermore, the possibility exists that our current infrared samples may be able to hydrolyze ATP in the absence of DNA. Fig.…”

Section: Atp Hydrolysis and Fluorescence Datamentioning

confidence: 99%

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Investigating Structural Changes Induced By Nucleotide Binding to RecA Using Difference FTIR

Butler

Hanchett

Rafailov

et al. 2002

Biophysical Journal

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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.

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Section: Infrared Difference Spectrasupporting

confidence: 66%

Section: Atp Hydrolysis Assaysmentioning

confidence: 99%

Section: Infrared Studiesmentioning

confidence: 99%

Section: Atp Hydrolysis and Fluorescence Datamentioning

confidence: 99%

Section: Atp Hydrolysis and Fluorescence Datamentioning

confidence: 99%

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Investigating Structural Changes Induced By Nucleotide Binding to RecA Using Difference FTIR

Butler

Hanchett

Rafailov

et al. 2002

Biophysical Journal

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Difference FTIR Studies Reveal Nitrogen-Containing Amino Acid Side Chains Are Involved in the Allosteric Regulation of RecA

2005

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The Escherichia coli RecA protein performs the DNA strand-exchange reaction utilized in both genetic recombination and DNA repair. The binding of nucleotides triggers conformational changes throughout the protein resulting in the RecA-ATP (high DNA affinity) and RecA-ADP (low DNA affinity) structures. Difference infrared spectroscopy has allowed us to study protein structural changes in RecA that occur after binding ADP or ATP. Experiments were performed on control and uniformly (15)N-labeled RecA in an effort to assign vibrational changes to protein structures and study the molecular changes associated with the allosteric regulation of RecA. Comparison of RecA-ATP and RecA-ADP data indicates that the protein adopts unique secondary structures in each form and altered N-H stretching vibrations in the RecA-ADP structure not observed in the RecA-ATP data. Numerous vibrations throughout the 1700-1300 cm(-)(1) region are influenced by isotopic substitution and imply that many nitrogen-containing side chains are altered after ADP binds to RecA. The RecA-ATP data contain unique vibrations that are not observed in the RecA-ADP data and may be associated with Gln, Lys, Arg, or Asn. Model compound studies on control and (15)N-labeled glutamine and lysine provide additional evidence that supports the tentative assignments of vibrations observed in our difference spectra. In addition, we provide evidence that nitrogen-containing amino acids are important in locking in the low-DNA affinity, more compact conformation of the protein and that some of these interactions may not be present in a more extended, flexible RecA-ATP conformation.

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Difference FT-IR Studies of Nucleotide Binding to the Recombination Protein RecA

Cited by 2 publications

References 33 publications

Investigating Structural Changes Induced By Nucleotide Binding to RecA Using Difference FTIR

Investigating Structural Changes Induced By Nucleotide Binding to RecA Using Difference FTIR

Difference FTIR Studies Reveal Nitrogen-Containing Amino Acid Side Chains Are Involved in the Allosteric Regulation of RecA

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