On The Water Subtraction In The Fourier Transform Infrared (FTIR) Spectra Of Proteins And Lipids

Therrien, Marie; Lafleur, Michel; Pézolet, Michel

doi:10.1117/12.970757

Cited by 1 publication

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“…Solvent H20 absorption was subtracted from DNA and SASP/DNA spectra by an interactive difference routine using the OBEY program. We used three criteria to establish correct solvent subtraction: (i) absence of the H20 association band at 2130 cm-1 (neither DNA nor SASPs absorb at this frequency, so this line is a suitable internal standard for solvent subtraction) (19), (ii) presence of a flat baseline in the 2000-to 1000-cm-1 range, and (iii) absence of negative side lobes. Enhancement and derivatization (using the Savitsky-Golay function) of spectra were performed using the CDS-3 programs.…”

Section: Methodsmentioning

confidence: 99%

Binding of small acid-soluble spore proteins from Bacillus subtilis changes the conformation of DNA from B to A.

Mohr

Sokolov²,

He³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

132

108

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Small acid-soluble spore proteins (SASPs) appear 3-4 hr after the onset of sporulation in Gram-positive bacteria and constitute up to 20% of the protein of mature spores. Previous studies using Bacillus subtlis deletion mutants lacking SASP-a and -1S have shown that such mutations abolish the elevated resistance ofspores to UV radiation. Analyses using circular dichroism and Fourier-transform infrared spectroscopy now demonstrate that bining a/f-type SASPs to DNA in vitro causes a structural change in DNA, from the B to the A conformation. This may provide the basis whereby a/f8-type SASPs confer increased spore UV resistance in vivo-by changing spore DNA conformation, they alter DNA photochemistry such that UV irradiation produces spore photoproduct instead of the more lethal cyclobutane-type thymine dimers.genes (termed ssp genes) exhibit a remarkably high degree of conservation of their primary sequences throughout evolution (10), and any one ofthe a/a-type SASPs appears capable ofconferring UV resistance on bacterial spores (11). By using several purified a/,a-type SASPs, we have examined the effects of SASP on DNA conformation, and we report here that SASP binding to DNA in vitro induces a B -> A conformation change. It seems likely that this phenomenon underlies the ability of SASP to protect spore DNA against thymine-dimer formation. Given their evolutionary antiquity (10), the SASPs also may have as-yet-undiscovered protein relatives that use the same strategy to protect cells against UV. The high intrinsic UV resistance of obligate anaerobic bacteria (12), for example, may reflect just such a system. Among the dramatic changes accompanying sporulation in Gram-positive bacteria, a major increase in resistance to killing by UV radiation ranks high as a factor ensuring that the organism will survive dormancy (1). Detailed study of this phenomenon has shown that UV irradiation of spores produces no detectable cis-syn cyclobutane-type thymine dimers in DNA-in sharp contrast with the predominance of this lesion in other UV-irradiated in vivo systems. Instead, the DNA of UV-irradiated spores accumulates primarily 5-thyminyl-5,6-dihydrothymine ("spore photoproduct") (2,3). Germinating spores contain a specific enzyme that efficiently repairs spore photoproduct (4), thereby ensuring a high probability of survival after exposure to UV.It is clear that DNA conformation can significantly alter photoreactivity (5) and the type of UV-induced photoproducts (6). Thus, one simple explanation for the change in DNA photochemistry that occurs during sporulation invokes an alteration in DNA conformation. Indeed, to explain the altered photochemistry of spore DNA, it was suggested a number of years ago that DNA in bacterial spores is in the A conformation whose stereochemical constraints inhibit thymine dimer formation (7). Howe-, er, the relationship between A-DNA and UV-induced formation of spore photoproduct has never been proven, and it has been suggested (6, 8) that hydration is a more important determinant of D...

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Section: Methodsmentioning

confidence: 99%