In partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biology

In the

School of Biological Sciences

Kasahun Neselu

Will defend his dissertation

STRUCTURAL STUDIES OF MEMBRANE PROTEINS BY CRYO-EM

Thursday, April 15th, 2021

2:00 PM

https://gatech.bluejeans.com/164266228

 Thesis Advisor:

Ingeborg Schmidt-Krey, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Committee Members:

Raquel Lieberman, Ph.D.

School of Chemistry & Biochemistry

Georgia Institute of Technology

Loren Williams, Ph.D.

School of Chemistry & Biochemistry

Georgia Institute of Technology

Yuhong Fan, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Matthew Torres, Ph.D.

School of Biological Sciences

Georgia Institute of Technology

Abstract

This study focuses on structural studies of two membrane proteins: signal peptide peptidase and photosystem II were studied by the electron cryo-microscopy (cryo-EM) approaches of 2D electron crystallography and single particle cryo-EM.

Signal peptide peptidase (SPP) is a small membrane protein of 33kDa and a member of the intramembrane aspartyl protease family. Several members of this protein family are medically relevant and are key drug targets. The small size of SPP makes X-ray crystallography and 2D electron crystallography suitable approaches for structural studies. Two-dimensional crystallization is a requirement for 2D electron crystallography. Optimization of 2D crystallization conditions for SPP, including the lipid-to-protein ratio (LPR), pH, temperature, and crystallization buffer, resulted in substantially improved SPP 2D crystals.

         Photosystem II (PSII) is a large membrane protein complex of 1.1 MDa found in photosynthetic organisms. PSII oxidizes water and generates oxygen. The size and sample heterogeneity of the active PSII makes single particle cryo-EM the most suitable approach for structure determination. Three samples were investigated that are relevant to conformational changes taking place during the photoinhibition repair mechanism of PSII, which involves replacement of the extrinsic subunits PsbO, PsbP, and PsbQ. The different complexes can be mimicked by subjecting the purified PSII to a salt wash, resulting in the removal of only the PsbP and PsbQ subunits, the “salt wash” sample, and a urea wash, resulting in the removal of the three extrinsic subunits, the “urea wash” sample. The active sample, with the extrinsic subunits in place, the salt wash sample, and the urea wash sample were subjected to extensive testing of cryo-EM grid preparation conditions to retain activity, maximize particle number, and optimize ice thickness. Image processing of cryo-EM data accounted for sample heterogeneity and resulted in three-dimensional reconstructions that clearly show the overall architecture of the 3 PSII samples.