PhD position in NMR-based structural enzymology combining NMR, SAXS and XFEL

SUNY Buffalo

Laboratory of Dr. Szyperski, Chemistry Department

BioXFEL STC funded project pursued in collaboration with

Drs. Edward Snell (HWI), Thomas Weiss (Stanford) and Marius Schmidt (UWM)

Research. Enzyme function is potentially linked to the entire protein conformational space sampled by enzyme and enzyme-intermediate complexes during catalysis. Importantly, this holds for lowly populated conformational sub-spaces of higher free energy, which can differ distinctly from the lowest free energy conformational sub-space typically captured in X-ray crystal and NMR structures. For large enzymatic ‘machines’, the most prominent example being the ribosome, FRET and cryo-EM have revealed the functional importance of large-scale conformational changes during substrate turnover. However, it is unknown to which extent large conformational changes associated with turnover typically exist for medium-sized enzymes, and if they are functionally important.

To address this question, our research focuses on the 29 kDa broad-spectrum
b-lactamase (BlaC) from Mycobacterium tuberculosis, a bacterial pathogen which causes worldwide ~1.5 million tuberculosis deaths each year. This enzyme is of outstanding biomedical importance because it conveys resistance [1] against b-lactam antibiotics (including the cephalosporin CEF) and greatly impedes antibiotic treatment of tuberculosis. Specifically, BlaCs hydrolyze b-lactam antibiotics and the detailed understanding of the enzymatic mechanism is of key importance to tackle antibiotic resistance.

This project focusses on the structural enzymology of BlaC combing new insights from solution nuclear magnetic resonance (NMR) spectroscopy, solution small angle X-ray scattering, and crystal X-ray scattering using free electron lasers (XFELs). Our unprecedented NMR and SAXS studies revealed the presence of large conformational excursions of the medium-sized BlaC during substrate turnover. In contrast, well resolved crystal structures were obtained for BlaC as well as catalytic intermediates in the pioneering Mix-and-Inject Serial (MISC) X-ray free electron laser (XFEL) studies of Schmidt and coworkers [1]. The central hypothesis driving our research is that the NMR-detected large excursions may well be of functional importance but are suppressed when the reaction proceeds in crystals. We received a grant for beamtime at the Stanford synchrotron to next perform time-resolved SAXS in order to (i) complement MISC and (ii) refine further structure-based models of the catalytic cycle.

Furthermore, we (i) characterized both competitive and non-competitive product inhibition by NMR chemical shift perturbation, and (ii) accurately measured BlaC Michaelis-Menten (MM) parameters and inhibition constants. To overcome limitations arising from fitting competitive and non-competitive inhibition from initial reaction velocities, we plan to fit our data using the integrated form of the Michaelis-Menten equation. Such fitting does not rely on the initial rate approximation and a form which considers simultaneous competitive and non-competitive inhibition is available [2]. We collaborate with Dr. Bezerra, who implemented the required non-linear fitting in the Solver Supplement of the Microsoft Office Excel program [3] and have tested this approach with our data.

Education and Training. This research project is exceptionally well-suited for cross-interdisciplinary training of PhD students in routine molecular biology for protein sample preparation, biophysics, NMR-based structural biology and advanced enzymology. This includes training in NMR, SAXS, and Xray crystallography, as well as circular dichroism and fluorescence spectroscopy molecular dynamics simulations

The successful candidate will be a PHD Student in the Chemistry Department of SUNY-Buffalo (UB) (https://arts-sciences.buffalo.edu/chemistry/graduate/overview/phd.html) and is expected to play an active role as a Teaching Assistant (TA). The student will be supported financially through the appointment as TA, research funding and possibly a scholarship.

Dr. Szyperski integrates novel research insights into his elective, special topic graduate course CHE 512 and organizes outreach biophysics webinars. The student will participate in our regular research meetings with collaborators, which includes meetings for our second NSF-funded project on the engineering of cold denaturing proteins.

Furthermore, the student will participate in the educational program of the BioXFEL STC (https://www.bioxfel.org/education), which was described by Dr. Bauer in a recent article [4], as a BioXFEL scholar (https://www.bioxfel.org/education/bioxfel-scholars/scholar-programs).

 

Interested students are welcome to send their CV along with a concise personal statement describing previous research experience and current research interests to Dr. Szyperski. He will then set up a Zoom meeting to become acquainted and to discuss opportunities.

 

Contact information

Thomas Szyperski

UB Distinguished Professor

Chemistry Department

University at Buffalo (UB)

This email address is being protected from spambots. You need JavaScript enabled to view it.

http://chemistry.buffalo.edu/people/szyperski

 

References

[1] Olmos, J.L. et al. Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography. BMC Biology, 2018, 16: 59.

[2] Bezerra, R. M. et al. Utilization of integrated Michaelis‐Menten equation to determine kinetic constants. Biochemistry and Molecular Biology Education 2007, 35: 145.

[3] Bezerra, R. M. et al. Utilization of integrated Michaelis–Menten equations for enzyme inhibition diagnosis and determination of kinetic constants using Solver supplement of Microsoft Office Excel. Computer Methods and Programs in Biomedicine 2013, 109, 26.

[4] Bauer, W. J., Woodruff, S. B. A science education model for large collaborative centers. Struct Dyn, 2021, 8: 020402.