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The effects of osmolytes and crowding on the pressure-induced dissociation and inactivation of dimeric LADH

DOI: 10.1039/C7CP08242H DOI Help

Authors: Karin Julius (TU Dortmund University) , Samy Ayoubi (TU Dortmund University) , Michael Paulus (TU Dortmund University) , Metin Tolan (TU Dortmund University) , Roland Winter (TU Dortmund University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Physical Chemistry Chemical Physics

State: Published (Approved)
Published: February 2018
Diamond Proposal Number(s): 14949

Abstract: Investigating the correlation between structure and activity of oligomeric enzymes at high pressure is essential for understanding intermolecular interactions and reactivity of proteins in cellulo of organisms thriving at extreme environmental conditions as well as for biotechnological applications, such as high-pressure enzymology. In a combined experimental effort employing small-angle X-ray scattering, FT-IR and fluorescence spectroscopy as well as stopped-flow enzyme kinetics in concert with high-pressure techniques, we reveal the pressure-induced conformational changes of the dimeric enzyme horse liver alcohol dehydrogenase (LADH) on the quaternary, secondary and tertiary structural level. Moreover, the effects of cosolutes and crowding agents, mimicking intracellular conditions, have been addressed. Our results show that beyond an increase of enzymatic activity at low pressures, loss of enzyme activity occurs around 600-800 bar, i.e. in a pressure regime where small conformational changes take place in the coenzyme's binding pocket, only. Whereas higher-order oligomers dissociate at low pressures, subunit dissociation of dimeric LADH takes place, depending on the solution conditions, between 2000 and 4000 bar, only. Oligomerization and subunit dissociation are modulated by cosolvents such as urea or trimethylamine-N-oxide as well as by the crowding agent polyethylene glycol, based on their tendency to bind to the protein's interface or act via their excluded volume effect, respectively.

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I22-Small angle scattering & Diffraction

Added On: 23/02/2018 09:47

Discipline Tags:

Physical Chemistry Biochemistry Chemistry Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)