원자 힘 현미경을 이용한 AKR2A와 Ribosome-Nascent Chain Complex간의 상호 작용 및 기능화된 Polythiophene 박막 연구

Abstract

Abstract

Chapter I. Single Molecule Force Spectroscopy for Biological and Materials Sciences Understanding mechanical processes at the single molecular level is a crucial aspect of both biology and materials science. In conventional measurement techniques on an ensemble or a bulk collection of molecules, the individual behavior of molecules cannot be distinguished, and only averaged characteristics can be measured. Single molecule force spectroscopy (SMFS) has been developed as a powerful technique to investigate the information about the behavior of an individual molecule for biological and materials systems. Using optical tweezers (OTs), magnetic tweezers (MTs), and atomic force microscopy (AFM), the interaction force between biomolecules can be directly quantified at the single-molecule level. Among the techniques, AFM is widely known for high resolution imaging of various structures and measuring single-molecular interactions with picoNewton force sensitivity and sub-nanometer positional accuracy. Also, AFM can be operated in liquid and under physiological condition, which has been employed to study the specific interaction of biomolecules and polymers at the single-molecule level. Furthermore, mapping capability of AFM can be applied for investigating the nanoscale distribution of recognition sites of biomolecules on surfaces and the variation of mechanical properties of polymer films.

Chapter II. Measurement of Interactions between AKR2A and Ribosome-Nascent Chain Complex by Atomic Force Microscopy In eukaryotic cells, organellar proteome biogenesis is pivotal for cellular function. Chloroplasts contain a complex proteome whose biogenesis includes posttranslational targeting of nucleus-encoded proteins to different suborganellar locations. However, the mechanism determining when and how nascent chloroplast-destined proteins are sorted in the cytosol is unknown. In this study, we investigated the question of when and how ankyrin repeat-containing protein 2 (AKR2A) specifically recognizes its nascent cargo proteins in the cytosol during protein targeting to chloroplasts. Binding behavior of AKR2A to ribosome-nascent chain complexes (RNCs) using AFM force measurements and mapping was studied at the single-molecule level. The study showed that the mode of interaction between AKR2A and RNCs may change depending on the stage of translation. Nascent AKR2A-client (OEP7) induces AKR2A binding to RPL23A of ribosomes before emergence from the exit tunnel. Subsequently, RPL23A-bound AKR2A binds to the targeting signal when it is exposed from ribosomes to initiate chloroplast targeting. Failure of AKR2A binding to RPL23A in planta severely disrupts protein targeting to the chloroplast outer membrane (COM). Thus, AKR2A-mediated targeting of COM proteins is coupled to their translation, which in turn is crucial for biogenesis of the entire chloroplast proteome.

Chapter III. Analysis of Functionalized Polythiophene Films by Atomic Force Microscopy AFM has been widely employed to characterize various properties of polymeric materials. Although copolymers that form self-assembled structures have attracted much attention, morphological maps created using AFM do not reveal the nanoscale structures of flat films. In this study, we report that force-extension curve-based analysis using anthracene-tethered AFM probes reveals the relevant behavior of functionalized poly(3,4-ethylenedioxythiophene) (PEDOT) copolymers. Functionalized ethylenedioxythiophene (EDOT) monomers, hydroxymethyl EDOT (EDOT-OH) and zwitterionic phosphorylcholine EDOT (EDOT-PC) were electropolymerized to prepare the homopolymers poly(EDOT-OH) and poly(EDOT-PC), and mixtures of these monomers were used to produce the copolymer poly(EDOT-OH)-co-poly(EDOT-PC). The PEDOT-OH film yielded force-extension curves for short stretching, and the PEDOT-PC film yielded curves for long stretching. A dendron-modified AFM tip with anthracene groups tethered at the end resulted in adhesion maps with the highest contrast. The analytical data for the copolymer films correlated with the corresponding monomer composition, and the maps revealed that the average size for the copolymer nanodomains ranged from 10–14 nm. Furthermore, PEDOT-PC obtained by potentiostatic electropolymerization and cyclic voltammetry at the highest scan rate showed adhesion maps with the highest ratio of pixels exhibiting long stretching curves. Additionally, the analysis showed that the relative ratio of pixels with short stretching curves was less sensitive to the polymerization method and conditions for PEDOT-OH. The analysis indicated that the thermal annealing of PEDOT-PC led to structural changes on the surface. These nanoscale changes were uniformly distributed. This approach can be applied to studies aimed at understanding the surface structure of other relevant polymers and copolymers at the nanoscale level.