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Nuclear Magnetic Resonance and Biochemical Investigations of Environmental Changes on Structure and Function of Subtilisins

  • Author(s): Liszka, Michael Joseph
  • Advisor(s): Clark, Douglas S
  • Reimer, Jeffrey A
  • et al.
Abstract

Many industrial processes used to produce chemicals and pharmaceuticals would benefit from enzymes that function under extreme conditions. Fortunately, bioprospecting for enzymes from extremophilic microorganisms has led to the discovery of new enzymes with high tolerance to non-natural conditions. However, bioprospecting is inherently limited by the diversity of environments present in nature. Protein engineering has also been a successful route to generate extremophilic enzymes by both rational mutagenesis and directed evolution. Ultimately, screening for activity for either type of enzyme under extreme conditions can be difficult. To continue development of new and more robust biocatalysts, there is increasing synergy between bioprospecting and protein engineering in developing extremophilic enzymes. Interesting areas considered include unnatural industrial conditions relevant to biocatalysis, biophysical properties of extremophilic enzymes, and industrially relevant extremophilic enzymes either found in nature or through protein engineering.

Decades of protein studies have used mutagenesis to alter enzyme function and rationalize the resulting effects with respect to the amino acid properties. In cases of bulk solvent changes, the exact effect on the enzyme and underlying mechanism for observed changes in enzyme function can be more difficult to decipher. We have used 1H-15N NMR titration experiments on the protease Subtilisin E for several altered solvent conditions to identify sub-molecular effects. Chemical shift and peak intensities were monitored as a function of temperature, dimethyl formamide (DMF) concentration and guamidinium HCl (GdmCl) concentration. The results reveal that the effects on the enzyme are specific to each perturbation consistent with unique sites identified by previous mutagenesis results. With the addition of 20% DMF, the most affected residues co-localize with previously reported mutation sites found from screening enzyme mutants in DMF. Temperature effects were observed as chemical shift perturbations in secondary structural elements. Guanidinum HCl addition deceased the intensity of residues from 156-179 also coincidental with a previous beneficial mutation site. Across all three changes, the ability of the enzyme to bind its substrate correlates with perturbations in the binding cleft. The NMR analysis presented here has provided insights into possible mechanisms for the beneficial mutations and has the potential to predict locations where mutagenesis may be most fruitful.

A retrospective analysis of mutation effects provides only a rearview understanding of the mechanism for the mutation's efficacy. To further investigate the structural basis for altered function, we subjected improved mutant proteins to the same analysis as the wild type enzyme. These data could provide a clearer picture of the changes imposed on the enzyme by the amino acid substitutions. Additionally, we compare the effects of mutations made to improve function in a specific environment with the mutated protein's general stability. NMR appears capable of identifying regions of an enzyme that are affected by a solvent, so we propose to use those regions as a guide to mutate the enzyme to improve its function.

To further to expand the use of enzymes in organic solvents, an expanded set of tools and techniques are required. Each of these tools provides a step toward understanding changes in enzyme function and improvements in enzyme design for use in organic solvents. The first tool is the de novo design and expression of an organic soluble protein. We were able to screen several generations of de novo designed proteins for their ability to express in aqueous conditions and attempted to transfer the expressed proteins to organic solvents. The last two techniques focus on developing techniques to work with enzymes in organic solvents. The first was born out of the need to improve screening enzyme libraries. Quantification of protein expression levels in cell-free mixtures is a difficult and necessary step for measuring the specific activity of a protein. Lastly, the measurement of spectral properties of subtilisin in organic solvents was an initial step toward high-resolution spectroscopy in organic solvents. I present informed outlooks for continuing or completing each of the projects.

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