Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae cryptochrome 1

Kathleen Sokolowsky, Maire Newton, Carlos Lucero, Bradley Wertheim, Jaryd Freedman, Frank Cortazar, Jennifer Czochor, Johannes Schelvis, Yvonne Gindt

Research output: Contribution to journalArticle

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Abstract

Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH-/FADH· reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH-/FADH· reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (KA ∼ 105 M -1 for photolyase and ∼104 M-1 for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed.

Original languageEnglish
Pages (from-to)7121-7130
Number of pages10
JournalJournal of Physical Chemistry B
Volume114
Issue number20
DOIs
StatePublished - 27 May 2010

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Cryptochromes
Deoxyribodipyrimidine Photo-Lyase
cholera
Escherichia
Escherichia coli
DNA
deoxyribonucleic acid
Thermodynamics
Calorimetry
Titration
thermodynamics
titration
Proteins
heat measurement
Adenosinetriphosphate
Substrates
thermodynamic cycles
proteins
Ultraviolet spectroscopy
Raman spectroscopy

Cite this

Sokolowsky, Kathleen ; Newton, Maire ; Lucero, Carlos ; Wertheim, Bradley ; Freedman, Jaryd ; Cortazar, Frank ; Czochor, Jennifer ; Schelvis, Johannes ; Gindt, Yvonne. / Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae cryptochrome 1. In: Journal of Physical Chemistry B. 2010 ; Vol. 114, No. 20. pp. 7121-7130.
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Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae cryptochrome 1. / Sokolowsky, Kathleen; Newton, Maire; Lucero, Carlos; Wertheim, Bradley; Freedman, Jaryd; Cortazar, Frank; Czochor, Jennifer; Schelvis, Johannes; Gindt, Yvonne.

In: Journal of Physical Chemistry B, Vol. 114, No. 20, 27.05.2010, p. 7121-7130.

Research output: Contribution to journalArticle

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