Dissertation
Investigating the role of protein dynamics using formate dehydrogenase as the model system
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2019
DOI: 10.17077/etd.005237
Abstract
Enzymes possess a diverse array of motions that operate at numerous timescales and lengths. They range from slow timescales such as seconds and milliseconds to fast timescales such as picoseconds and femtoseconds. The importance of enzyme motions (dynamics) has been under constant debate for decades. One of the factors contributing to the lack of knowledge on enzyme motions is the difficulty in understanding their role in enzyme function, which requires tailored approaches. Computational and theoretical, as well as certain experimental techniques have been utilized to perceive the task of slow enzyme dynamics. On the contrary, it has been quite challenging to assess the importance of fast enzyme dynamics due to the scarcity of suitable experimental techniques and accessible enzyme systems.
This work is primarily focused on using formate dehydrogenase (FDH) from Candida boidinii to investigate the role of femtosecond-picosecond enzyme dynamics in catalysis. FDH catalyzes hydride transfer from formate anion to NAD+. Two types of alterations, namely, mass-modulation and mutagenesis were performed on FDH. Kinetic isotope effect studies (KIEs) were employed to investigate the effect of these alterations on the chemical step (C-H→C transfer). Mass-modulation by isotopic labeling of non-exchangeable hydrogen (H), carbon (C) and nitrogen (N) atoms in the enzyme with their heavy isotopes perturbs the whole array of enzyme dynamics to varying degrees. Isotopic labeling studies not only suggest a link between fast enzyme dynamics and catalysis but also raise intriguing concerns about the use of isotopes in techniques such as nuclear magnetic resonance (NMR), X-ray crystallography, neutron scattering, and neutron protein crystallography. Altering the donor-acceptor distance by introducing active-site mutations in FDH unravels a correlation between C-H→C transfer and enzyme dynamics at its transition state.
In addition to KIEs, two-dimensional infrared spectroscopy (2D IR) was performed on the heavy isotopologues of FDH. 2D IR is a technique that measures the vibrational relaxation of a competitive inhibitor, azide anion bound to the active site of FDH that yields dynamics information on the active site environment for an analogous transition state structure. The results from spectroscopic and kinetic experiments reveal diverse effects caused by mass-modulation and mutagenesis of FDH.
Details
- Title: Subtitle
- Investigating the role of protein dynamics using formate dehydrogenase as the model system
- Creators
- Chethya Upalakshi Ranasinghe
- Contributors
- Christopher M Cheatum (Advisor)Amnon Kohen (Advisor) - University of Iowa, ChemistryDaniel M Quinn (Committee Member)Alexei V Tivanski (Committee Member)Tori Z Forbes (Committee Member)Ernesto Fuentes (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Chemistry
- Date degree season
- Autumn 2019
- DOI
- 10.17077/etd.005237
- Publisher
- University of Iowa
- Number of pages
- xv, 88 pages
- Copyright
- Copyright 2019 Chethya Upalakshi Ranasinghe
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 80-88)
- Public Abstract (ETD)
Enzymes (and proteins) play vital roles in the maintenance of life by catalyzing various biological reactions. This thesis research discusses how enzymes work with a focus on how different motions (dynamics) within enzymes contribute to their function. Comprehending the link between enzyme dynamics and function is important in designing new therapeutic drugs, inhibitors and engineered enzymes with novel functionalities. Changing the local structure by mutagenesis or mass-modulation by isotopic labeling leads to perturbation of the protein dynamics, thus allowing the investigation of their significance in protein function.
Formate dehydrogenase (FDH) is used as the model system for the experiments discussed in this thesis. Although FDH is non-existent in humans, it acts as a suitable model system for our kinetic and spectroscopic studies in establishing a link between protein dynamics and function. Studies on mutagenesis and mass-modulation provide insight into how femtosecond-picosecond motions (fast vibrations) in the enzyme dictate its main functionality.
Mass-modulation further sheds light on the effects of isotopic labeling in proteins that need to be taken into consideration when employing structure determination techniques such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, neutron scattering, etc. The studies open more avenues of research to further explore the complex mechanism of these fascinating molecular machineries.
- Academic Unit
- Chemistry
- Record Identifier
- 9983779798602771
Metrics
33 File views/ downloads
46 Record Views