Master's lecture in biochemistry -Arnór Freyr Sævarsson

Master's student: Arnór Freyr Sævarsson
Title: Thermostabilization of VPR, a cold adapted subtilase, by proline substitutions into surface loops and monitoring the dynamics of the active site through electron paramagnetic resonance
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Faculty: Faculty of Physical Sciences
Advisor: Magnús Már Kristjánsson, Professor
Examiner: Ólafur Þór Magnússon, deCode

Abstract

"The first goal of this project was to understand the thermostabilization effect of prolines in surface loops of VPR a cold adapted serine protease from Vibrio sp. (PA-44). The mutations
N3P, I5P, N238P, and T265P were introduced into VPR based on the location of the prolines found at corresponding sites in surface loops of aqualysin I (AQUI), a thermophilic structural homologue from Thermus aquaticus. The single variants N3P, I5P showed most increase in stability while the T265P and N238P variants had mild increase and decrease in stability respectively compared to the pseudo wild type VPRΔC. The double (N3P/I5P), triple
(N3P/I5P/N238P and N3P/I5P/T265P), and quadruple variant (N3P/I5P/N238P/T265P) were all more stable than if the stability effects of the individual proline substitutions were added together. The most stable variants were N3P/I5P/T265P and N3P/I5P/N238P/T265P
with around 8-9°C increase in stability compared to VPRΔC according to TM and T50%. The thermostabilization of the proline substitutions conferred to VPR were therefore additive.
All the proline mutations caused minimal to no effect on the activity characteristics of VPR.

The second goal of this project was to reveal the possible relationship between molecular flexibility and catalytic activity in the cold-active VPR and the thermostable AQUI. This was achieved with a chemical modification of the active-site serine to cysteine followed by spin labeling. The motion of the active-site spin label was then monitored with electron paramagnetic resonance (EPR). The EPR line shape and scaled mobility (Ms) of the spin labeled VPRΔC and AQUI wt reflected that the motion of the spin label was largely similar in both enzymes and immobile. This might suggest that the motion of the active site in the
near vicinity to the active-site serine, which had been modified to cysteine, is similar for VPRΔC and AQUI wt. This is against the general notion that enzymes from psychrophiles achieve their cold activity through increased flexibility of the active site. AQUI D98S, which has increased turnover rate (kcat) and substrate affinity (KM), exhibited considerably more motion of the active-site spin label in comparison to AQUI wt. This suggests that there might
be increased localized motion or possibly some alteration to the active site in AQUI D98S compared to AQUI wt."