Multi-domain Protein Unfolding Pathway Studies by Single Molecule Techniques

Large multi-domain proteins, which are ubiquitous in the proteomes in eukaryotic and prokaryotic organisms, still lack intensive studies on their folding mechanisms due to their complicated interactions between and inside of their domains. My work is broadly aimed at characterize folding behaviors and mechanisms of large, multi-domain proteins.

In the first part of my thesis work, I developed a novel mechanical folding polypeptide probe based on the anti-parallel coiled-coil domain of a natural protein (Archeal Box C/D sRNP Core Protein) to enable us to capture the progress of the unfolding front along the host protein structure. In this way, the structural origin of the signal from single molecule force spectroscopy (SMFS) based on atomic force microscopy (AFM) can be directly identified. Beyond this published work, I also characterized and compared the unfolding pathway of two homologous multi-domain proteins – yeast phosphoglycerate kinase (PGK) and its E.coli homolog. I found that yeast PGK has much higher mechanical stability than E.coli PGK although previous literature reported that they had similar thermodynamics stability determined by bulk measurements. In collaboration with Mr. Zackary N. Scholl, we characterized another multi-domain protein, protein S, and its two isolated domains (protein S N terminal domain and C terminal domain). By matching the statistical distributions of the unfolding forces from the truncated domains with the distributions of forces from full protein S, we solved the problem of assigning force peaks in the force-extension (FE) curves to individual domains. However, accurate determination of the structural correspondence to the force peaks still needs insertion of CC probes into the loop regions of these multi-domain proteins.

In the second part of my thesis work, I focused on integrating our AFM-SMFS with fluorescence microscopy methods. In particular, I have focused on constructing and mounting a home-made AFM force spectrometer on a high magnification inverted microscope to monitor fluorescence signal changes when stretching a single protein molecule. I am also setting up a total internal reflection fluorescence microscope (TIRF) for combination of Förster resonance energy transfer (FRET) and SMFS measurements in the future.

In Chapter 1, I will discuss background of multi-protein unfolding, SMFS method principles and coarse-grained (CG) steered molecular dynamics (SMD) simulation. This chapter will focus on the importance of understanding multi-protein unfolding pathway, the advantage of using SMFS as an experimental way of characterizing multi-domain protein unfolding behavior. As a theoretical way to analyze what event happened during the forced unfolding of multi-domain protein, CG-SMD simulations usually support our SMFS results, and providing details for interpreting FE curves obtained in SMFS measurements.

In Chapter 2, I present my recently completed work on developing a mechanical force probe based on an anti-parallel coiled-coil polypeptide chain. This work has just been published by Angewandte Chemie International Edition. The probe I developed provides a new way to determine the structural relation of the peaks shown in protein unfolding FE curves.

In Chapter 3, I report my recently started work on characterization of yeast PGK. I will show the preliminary data we obtained, (this work is also in collaboration with Mr. Zackary N. Scholl), and discuss differences in the FE traces of the two homologs and propose future work directions.

In Chapter 4, I briefly mention another work, Ca2+dependence of protein S unfolding pathway. The NPS and CPS domains were also individually studied by AFM, and their unfolding data statistics helped exploring comprehensively the structural origin of full protein S unfolding data.

In Chapter 5, I will introduce my completed work on combination of AFM and inverted Zeiss microscope. The completion of this work will enable simultaneous recording of the fluorescence signal in an SMFS experiment performed on a single molecule in the future.

In Chapter 6, I will describe my recent work on building a TIRF microscope to realize TPM measurement on single molecule multi-domain protein in our lab. We hope that the integrated instrument could enable us to detect FRET signal coming from unzipping of the CC probe when stretching by AFM.