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Temperature adaptation in Gillichthys (Teleost: Gobiidae) A₄-lactate dehydrogenases : identical primary structures produce subtly different conformations

Peter A. Fields^1,*, Yong-Sung Kim², John F. Carpenter² and George N. Somero¹

¹ Hopkins Marine Station, Biological Sciences Department, Stanford University, Pacific Grove, CA 93950, USA
² School of Pharmacy, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA

View larger version (20K): [in a new window]   Fig. 1. (A) Buffer-corrected tryptophan fluorescence spectra of Gillichthys mirabilis and G. seta muscle-type lactate dehydrogenases (A4-LDHs) at 20 °C. Excitation wavelength was 295 nm. (B) Thermal denaturation profiles of the A4-LDH forms monitored using tryptophan fluorescence (excitation wavelength 295 nm, emission wavelength 377 nm). Curves were fitted to the data as described in the text. Gillichthys mirabilis A4-LDH Tm=58.4±0.1 °C and G. seta A4-LDH Tm=55.5±0.1 °C.

View larger version (23K): [in a new window]   Fig. 2. (A) Buffer-corrected far-ultraviolet circular dichroism (CD) spectra of Gillichthys mirabilis and G. seta muscle-type lactate dehydrogenases (A4-LDHs) at 20 and 40 °C. (B) Thermal denaturation profiles of the A4-LDH forms monitored using CD spectroscopy; absorbance was measured at 222 nm. Curves were fitted as described in the text; G. mirabilis A4-LDH Tm=60.2±0.1 °C and G. seta A4-LDH Tm=58.4±0.1 °C). MRW, residue ellipticity.

View larger version (26K): [in a new window]   Fig. 3. (A) Near-ultraviolet circular dichroism (CD) spectra of Gillichthys mirabilis and G. seta muscle-type lactate dehydrogenases (A4-LDHs) at 20 °C, and the subtraction spectrum (G. mirabilis minus G. seta; MIR minus SETA). (B) Second-derivative absorbance spectra of the two forms at 20 °C and the subtraction spectrum. The peak-to-trough distances, a and b, used to calculate the ratio r are illustrated. Note the difference in x-axis range between the two panels. Mir, to be defined; SETA, to be defined; MRW, residue ellipticity; d2A/dx2, second derivative of the spectrum.

View larger version (25K): [in a new window]   Fig. 4. Change in the ratio of amide II to amide I peaks over time during hydrogen/deuterium exchange monitored by infrared spectroscopy. Symbols represent data collected, lines represent the best fit of a double-exponential decay to each experiment, as described in the text.

View larger version (20K): [in a new window]   Fig. 5. Inverted second-derivative infrared spectra of muscle-type lactate dehydrogenase (A4-LDH) across the amide I region. Each panel shows spectra 2, 7, 12, 17, 25, 40 and 60 min after addition of protein to 75% D2O buffer. Peaks in the spectra correspond to specific secondary structures, and the arrows at approximately 1655 cm-1 (-helix) and approximately 1639 cm-1 (ß-sheet) indicate the direction of change in peak height over time. d2A/dx2, to be defined.

View larger version (25K): [in a new window]   Fig. 6. (A) Change in height of peaks at approximately 1639 cm-1 (ß-sheet structure) in Gillichthys muscle-type lactate dehydrogenases (A4-LDHs) inverted second-derivative infrared spectra (see Fig. 5) from 2 min to 360 min (20°C) or 300 min (40°C) after addition to 75% D2O. (B) Change in height of peaks at approximately 1655 cm-1 (-helical structure) over time.

View larger version (51K): [in a new window]   Fig. 7. (A) Gillichthys muscle-type lactate dehydrogenase (A4-LDH) monomer with Tyr residues (gray wire-frame) and `major mover' (dark gray ribbon) secondary structures labeled. Substrate and cofactor enter the active site through the opening to the left among the major movers, which close down to form the catalytic vacuole. (B) LDH-A dimer, showing major movers, as in A, and the positions of Tyr246 residues hydrogen-bonded to a trapped water molecule (gray sphere) in the intersubunit contact area. Helix 3G is shown as a white ribbon below helix 1G—2G and Tyr246. Structures were based on the homology model of dogfish A4-LDH (Abad-Zapatero et al., 1987) and pig A4-LDH (Dunn et al., 1991) produced using the SWISS-PROT program (Guex and Peitsch, 1997) and visualized using Rasmol.