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Leptospira biflexa

Leptospira (Greek leptos, "fine, thin" and Latin spira, "coil")[1] is a genus of spirochaete bacteria, including a small number of pathogenic and saprophytic species.[2] Leptospira was first observed in 1907 in kidney tissue slices of a leptospirosis victim who was described as having died of "yellow fever."[3]

Wild Type

  • \( \lambda_{max} = 457\; nm\),
  • Recovery time constant: < 1 sec,
  • Abs 457 nm = 211.216 OD (measurement was done for 400 fold diluted sampme),
  • You will dilute 40-fold to get 0.5 od in the 1mm light path.
  • ε (457nm) = 47,100,
  • Purity index = 0.50,
  • 10 μl in each tube (two vials sent)
  • spectra data (Ascii)

HhalWT pB absorbs at 353 nm, and the LeptWT pB maximum is 392 nm. The small peak at 494 nm in LeptWT is pR, which is in the equilibrium between pR and pB. In LeptPYP as well as SalinibacterPYP (and R.centenumPYP), the equilibrium is shifted to pR resulting in appearing pR, even under the neutral pH. In contrast, the pR appears higher pH (> 10) in HhalPYP. In the HhalWT, small peak appears at around 450 nm but it is not pR but a noise due to the fluctuation of baseline in the original spectra.

Red: HhalWT pB spectrum, Blue: LeptWT pB spectrum. The spectra were produced by the calculation: (PYP-Light) – (PYP-dark) x (factor). Where (factor) is the value that the contribution of (PYP-dark) in (PYP-Light) would be cancelled. The spectra were normalized at 278 nm. pH was 7.5

The measurement was kinetic mode with continuous illumination. I used HP spectrophotometer so I took whole spectra (250 nm -550 nm) during kinetic measurement. As the spectra were noisy, dark or light spectrum used for the subtraction is averaged one.

I68P mutant

I68P mutant of Leptospira biflexa PYP, pH 7.5, in 10mM Tris-HCl buffer.

  • \(\lambda_{max} = 459\; nm\),
  • Photocycle recovery time constant: < 1 sec,
  • Abs459nm = 237.62 OD (measurement was done for 400 fold diluted sampme),
  • ε (459nm) = 51400,
  • Purity index = 0.46,
  • 20 μl in each tube
  • spectra data (Ascii)

 

Absorption spectra of HhalWT (446 nm), LeptWT (457 nm), LeptA50T (452 nm) and LeptI68P (459 nm).

 


The reference for the red-shift in Hhal T50V PYP is: Mihara K, Hisatomo O, Imamoto Y, Kataoka M, Tokunaga F. 1997. Functional expression and site-directed mutagenesis of photoactive yellow protein. J. Biochem. 121, 876-880.