Abstract
The developments of atomic force microscopy (AFM) are growing up in recently years. The advantages of AFM, such as high resolution at subnano level, application on living cell, and the employment of protein-protein interaction, which make the AFM easily and quickly ultilized on life science. Application of AFM on biological sample could provide new methodology for life science. In this thesis, we applied AFM to plant vacuole, membrane protein investigation and protein-protein interactiona and further provided new insights into these areas. The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. Higher plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, both pumps respond to stress differentially in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings (Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. Albeit, no significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent Vmax but not in KM values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is accordingly proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts. In the second part, we took vacuolar proton-translocating pyrophosphatase (V-PPase) as the model system for membrane protein study. V-PPase generates a proton electrochemical gradient across the membrane by hydrolyzing pyrophosphate for maintenance of acidic condition of vacuoles and translocation of secondary metabolites, ions, and even toxics. The enzymatic activity of V-PPase could be stimulated by relatively high concentration of K+, but inhibited by F-, Na+, Ca2+ and excess PPi. In this study, we used the yeast expression system to express hexa-histidine tagged mung bean V-PPase and employed detergent n-dodecyl 刍-D-maltoside (DDM) to solubilize the protein from microsomal membrane, followed by a Ni2+-nitrilotriacetate (Ni2+-NTA) affinity column to yield a highly purified enzyme. The specific activity of purified His-tagged V-PPase was approximately 86.4 ± 7.4 μmol PPi /mg.h, at least 6.5 fold purification compared to that on the vesicle membrane. The specific activity of His-tagged purified V-PPase were approximately 59% compare to the mung bean innate one. Further characterization indicates that the His-tagged V-PPase thus obtained resembles primarily those on membrane in most enzymatic features. The spectroscopic analyses including circular dichroism spectroscopy on His-tagged V-PPase revealed variations in conformational change induced by ions, as those inhibitors Na+, Ca2+, and F-, of this proton translocase. These results confirm the effect of ions are exerted concomitantly with the conformational (secondary structural) changes. The AFM technique uses a tiny stylus on a cantilever that is dragged across the lipid layer surface, and the deflections recorded are used to map the surface topology. Furthermore, the tiny stylus can be used as a fishing pole to fish the membrane protein out of lipid bilayer for determining the force barriers of TMs-TMs of TMs lipid interaction. For this purpose, we reconstituted V-PPase into lipid bilayer in the same orientation to minimize unexpected results when it is poured off the membrane by AFM stylus. The force extension curves thus reflect the mechanical stability of TMDs and interactes with vicinitic TMDs or lipid bilayers. In the presence of PPi and its analog, IDP, the force extension curves revealed significantly changes occurred on the putative PPi binding sites. The conformational changes were also confirmed using other spectroscopes such as Circular Dichroism (CD). Taken together, we proposed a working model for the PPi hydrolysis showing an interaction between domains upon binding of substrate and its analogs. In the third part, we used AFM to investigate the protein-protein interaction, especially the pathogenic protein and host cell interaction. We took Leptospira outer membrane lipoprotein as the model system to explore the protein-protein interaction mechanism. Leptopirosis is a renal disease caused by pathogenic Leptospira that primarily infects the renal proximal tubules, consequently resulting in severe tubular injuries and malfunctions. The protein extracted from outer membrane of this pathogenic strain contains a major component of a 32-kD lipoprotein (LipL32), which is absent in the counter membrane of nonpathogenic strains and is implicated as a crucial factor for host cell infection. Previous studies showed that LipL32 induced inflammatory responses as well as interacted with extracellular matrix (ECM) of the host cell. However, the exact relationship between LipL32 mediated inflammatory responses and ECM binding is still unknown. In this study, atomic force microscope (AFM) with its tip modified by purified LipL32 was used to determine the interaction between LipL32 and cell surface receptors. Furthermore, an antibody neutralization technique was employed to identify Toll-like receptor 2 (TLR2) but not TLR4 as the major target of LipL32 attack. The interaction force between LipL32 and TLR2 was measured as approximately 59.5 ± 8.7 pico-newton (pN), concurring with the theoretical value for a single pair molecular interaction. Moreover, transformation of TLRs deficient cell line with human TLR2 brought the interaction force from basal level to approximately 60.4 ± 11.5 pN, confirming unambiguously TLR2 as counter receptor for LipL32. The stimulation of CXCL8/IL-8 expression by full-length LipL32 protein as compared to that without N-terminal signal peptide domain suggests a significant role of the signal peptide of the protein in the inflammatory responses. This study provides direct evidence that LipL32 binds to TLR2, but not TLR4, on cell surface and a possible virulent mechanism for Leptospirosis is accordingly proposed.