Kinetic stability and temperature adaptation. Observations from a cold adapted subtilisin-like serine protease.

Life on earth is found everywhere where water is found, meaning that life has adapted to extremely varied environments. Thus, protein structures must adapt to a myriad of environmental stressors while maintaining their functional forms. In the case of enzymes, temperature is one of the main evolutionary pressures, affecting both the stability of the structure and the rate of catalysis. One of the solutions Nature has come up with to maintain activity and stability in harsh environments over biological relevant timescales, are kinetically stable proteins. This thesis will outline work carried out on the kinetically stable VPR, a cold active subtilisin-like serine protease and discuss our current understanding of protein kinetic stability, temperature adaptation and our current hypothesis of the molecular interactions contributing to the stability of VPR. The research model that we have used to study these attributes consists of the cold active VPR and its thermostable structural homolog AQUI. The results discussed in this thesis will be on the importance of calcium, the role of prolines in loops, the role of a conserved N-terminal tryptophan residue and lastly primary observations on differences in active site dynamics between VPR and AQUI. A model is proposed of a native structure that unfolds in a highly cooperative manner. This cooperativity can be disrupted, however, by modifying calcium binding of the protein or via mutations that affect how the N-terminus interacts with the rest of the protein. The N-terminus likely acts as a kinetic lock that infers stability to the rest of the structure through many different interactions. Some of these interactions may be strengthened via proline residues, that seemingly act as anchor points that tend to maintain correct orientation between these parts of the protein as thermal energy is increased in the system. Our results give a deeper insight into the nature of the kinetic stability, the importance of cooperativity during unfolding of kinetically stable proteases, synergy between distant parts of the protein through proline mutations and how different calcium binding sites have vastly differing roles. The results provide a solid ground for continuing work in designing enzyme variants with desired stabilities and activities and improve our understanding of kinetically stable systems.The Icelandic Research Fund [grant number 162977-051

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Publisher's version (útgefin grein)Protein stability is a widely studied topic, there are still aspects however that need addressing. In this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VPR, using the thermostable structural homologue AQUI as a template. Four locations for proline substitutions were chosen to imitate the structure of AQUI. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPRΔC_N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. In addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.This work was supported by The Icelandic Research Fund [Grant Number 162977-051].Peer Reviewe

The effects of selected proteinase inhibitors on the activity of subilases from psychrotrophic, mesophilic and thermophilic microorganisms

The interactions between proteases and protease inhibitors is an important part of maintaining balance in organisms, without inhibitors proteases would degrade various components and parts of the organism and cause damage and even death. These protease-protease inhibitor interactions are also good models for studying protein-protein interactions and therefore give clues to the characteristics of the chosen protein. In this study which is a part of a larger research project on temperature adaptation of subtilisin-like serine proteinases, or subtilases, three such homologous enzymes from the proteinase K family were studied with regard to inhibition effects by various inhibitors on their activities. The subtilases where a cold adapted protease from a psychrophilic bacterium of a Vibrio species (VPR), proteinase K from the mesophilic fungus Engyodontium album (PRK) and aqualysin I from the thermophilic bacterium Thermus aquaticus (AQUI). A comparative research was carried out on these proteases with respect to inhibition by nine different inhibitors. Based on the observed inhibitory pattern on the enzymes three of these inhibitors were selected for further measurements. These inhibitors were turkey ovomucoid trypsin inhibitor (TOM) and the two synthetic inhibitors, chymostatin (CHYS) and phenylmethylsulfonyl fluoride (PMSF). In order to determine difference in inhibition patterns, the kinetic rate constants were found from activity measurements of the enzymes in the absence and presence of the inhibitors. The hypothesis was that the cold adapted protease would be inhibited at the fastest rate as they have greater molecular flexibility and presumeably more accessibility of the active site residues. In fact the opposite was indeed observed as the observed inhibition rates were fastest in the case of the thermophilic AQUI. From the measurements done with PMSF against the subtilases it was determined that a possible governing factor causing the slower rate of inhibition of PRK and VPRΔC is the larger entropic contribution to the activation barrier for inhibition, most likely as the result of larger structural flexibility of these two subtilases in the uninhibited form compared to the thermophilic AQUI

Rational design of the cold active subtilisin-like serine protease VPR towards higher activity and thermostability

This research project builds on research previously done on the subtilisin-like serine proteinase VPR, from a psychrotrophic Vibrio species and its structural homologue aqualysin I (AQUI) from the thermophile Thermus aquaticus. We set out to design a mutant of VPR using site directed mutagenesis that would be both more stable against heat denaturation and retain the high activity of the wild type enzyme. Starting with two different templates, one being a C-terminal truncated form of the enzyme (VPRΔC), containing two proline mutations, N3P/I5P, close to the N-terminus of the protein, which had shown increased stability but loss of catalytic activity. The ∆C truncated form was produced by introducing a mutation as a stop codon at C277 to imitate the structure of AQUI in more detail. The other template contained the ∆C mutation and additional six mutations on a loop that may act as a hinge for movements that are postulated to be important for catalysis. The A116T/Q117R/A119H/S120R/G121R/S123A (6x) mutant had shown an increase in activity without losing stability to any degree. On top of these templates two mutations were added; N15D and Q142K. The N15D mutation had been shown to introduce a salt bridge yielding higher stability but with no detrimental effects on activity. The Q142K exchange on the other hand increased significantly the catalytic activity of the enzyme. Thus, we attempted to improve stability of the two mutants by introducing the N15D mutation, while the Q142K mutations was added with the purpose of increasing catalytic activity. The VPR∆C/N3P/I5P/N15D/Q142K mutant was a success, giving an 8°C rise in the Tm, a 10°C rise to the T50% and the catalytic activity was slightly higher than that of the wild type enzyme. The VPR∆C/6x/N15D/Q142K led to a 3°C rise in both Tm and T50%. To examine the effects of the N3P/I5P mutation on the flexibility of the structure, fluorescence quenching with acrylamide was preformed comparing AQUI, VPR∆C and VPR∆C/N3P/I5P. The results indicated that the environment of Trp6 in VPR∆C/N3P/I5P is not as accessible as in VPR∆C probably due to tighter packing of the N-terminus.Rannsóknasjóður Háskóla Ísland