PT1 Thermodynamic Characterization of DNA-binding Activity of Human Lon Protease

Si-Han Chena, Shih-Hsiung Wu, Ph.D.a,b.

aInstitute of Biochemical Science, National Taiwan University, Taipei city, Taiwan Republic of China,
bInstitute of Biochemistry, Academia Sinica, Taipei city, Taiwan Republic of China.

 

Lon is a class of ATP-dependent protease, which is committed to several crucial functions including proteolytic, ATPase, chaperone, and DNA-binding activities. Previously, human Lon protease (huLon) has been reported to specifically bind an element of antisense light strand promoter (24-mer LSPas) within the displacement loop (D-loop) region of mitochondrial genome. In this study, the potential binding sequence for huLon containing six consecutive guanine bases, 24-mer LSPas, was found to form G-quadruplex in the electrophoretic and circular dichroism spectroscopic analysis. We have investigated the thermodynamics of the binding reactions of huLon to 24-mer LSPas and 8-mer G-quartet-forming unit (TG6T) respectively by using isothermal titration calorimetry. The interactions between huLon and 24-mer LSPas were primarily driven by enthalpy change, which showed strong dependence on temperature, rendering a large negative reaction heat capacity, -607.82ˇÓ31.39 cal∙mol-1∙K-1. Though the binding of 8-mer G-quartet-forming unit to huLon was quite comparable in binding constant and free energy change with that of 24-mer LSPas, the former was mainly entropically driven, and associated with a negligible change in heat capacity, 67.29ˇÓ4.52 cal∙mol-1∙K-1, which reflected a lack of strong enthalpy-entropy compensation, in contrast to what was observed in the binding reaction of 24-mer LSPas. The results indicated that huLon not only interacted with the specific site but also bound to the element of G-quartet-forming unit within this specific sequence with appreciable affinity. We suggest that the polyanionic structure of G-quadruplex with high charge density might play an important role in conducting huLon to a specific binding site. The energetic interpretations may shed light on the DNA-binding mechanism of human Lon protease.

 

 

PT2 Solution structure of proteins probed by the small-angle X-ray scattering instrument newly installed at the NSRRC

Yu-Shan Huanga, U-Ser Jeng*a, Ying-Huang Laia, Ya-Sen Suna , Ming-Tao Leea , Keng S. Lianga , Jhih-Min Linb, and Tsang-Lang Linb

aNational Synchrotron Radiation Research Center, 101 Hsin-Ann Road, Hsinchu Science Park, Hsinchu 30076,Taiwan;
bDepartment of Engineering and System Science, National Tsing Hua University, Hsinchu 30013, Taiwan

 

A small-angle X-ray scattering (SAXS) instrument was recently installed at the wiggler beamline BL17B3 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. With a flux of 1010 - 1011 photon/s at the sample in the energy range of 5 to 14 keV, the instrument is capable of probing protein conformation in solution at a concentration of few mg/ml. Structural information, including size and shape as well as the aggregation number of proteins in solution can be deduced from SAXS profile analysis. With a water-bath for the sample holder, influence of temperature on protein unfolding, aggregation, or protein binding can be monitored between -10˘J to 150˘J. Peptides embedded in multilamellar lipid vesicles can be traced from the SAXS data obtained. Furthermore, for systems of lipid membranes invaded by peptides, the grazing incidence SAXS performed on the instrument can provide a way to study the peptide structure in the in-plane of the membrane as well as the correlation of peptides across the lipid bilayers of the host membranes. As an example, we report the structure evolution of lysozyme during an unfolding process induced by temperature. The Guinier approximation for the SAXS data measured reveals that lysozyme in aqueous solution has a stable globular shape with a radius of gyration Rg of 15.5 Å in the temperature range 30-50˘J. The size (Rg value) increases sharply at 60˘J and reaches 25.5 Å at 70˘J. Model simulation for the SAXS data show that during the unfolding process, lysozyme transforms from a globular shape to an elongated ellipsoid-like shape. Using a program "CRYSOL" developed by Sergun et al. to fit the SAXS data with an averaged molecular envelope based on the pdb data of the protein, we illustrate the evolution of lysozyme conformation during the unfolding process.

 

 

PT3 Towards full realization of near-atomic structural determination of single macromolecules by cryo microscopy: a study on the effect of phase contrast

Tsung-Kai Chiu, Chi-fu Yen, Cheng-wei Wu, Wei-hau Chang

Institute of Chemistry, Academia Sinica, Taipei, Taiwan.

 

Recently, low-temperature electron microscopy (cyo-EM) has emerged as a powerful tool for visualizing individual unstained cells and biological macromolecular assemblies, or ˇ§single particlesˇ¨, in rapidly frozen thin films of amorphous ice. Specifically, many biologically highly relevant proteins participate as members in large macromolecular complexes in the cell, whose structural analyses are not available for crystallography, can be determined by single particle reconstruction from cryo-EM images. Henderson had argued that it would be possible to determine atomic structure of a protein regardless of its size if sufficient particle images are coherently averaged. Under current experimental conditions, we still fall short of such promise, which motivated us to investigate the causes systematically. Among many limiting factors such as molecular heterogeneity, specimen drift and charging, contrast, which is known as a controllable parameter and a crucial contributor to image quality, has not been clearly addressed. In single particles structural analyses, three steps are required: particle segmentation, particle classification and particle alignment. We thus investigated how the efficiency of these steps were affected by contrast, by using simulated images to decouple influences from other factors in order to quantify the performances of each step under different microscopy contrast conditions, including conventional phase contrast and Zernike phase contrast. Our results substantiate that phase contrast dominates the efficiency of image segmentation, and also influences the performance of classification, especially when the contrast to noise ratio is low. As for the alignment, though larger defocus provides better contrast with conventional electron microscopy, we discovered the associated oscillation of high frequency degrades the coherence of alignment. We thus conclude Zernike mechanism would be helpful in extending the power of single particle imaging by cryo-EM to higher resolution because it can provide high contrast even at close to focus condition.

 

 

PT4 Molecular mechanism of focal adhesion kinase activation via phosphoinositides

Tang-Long Shen*, Hui-Yuan Tseng*, Yu-Chin Lin#, Yi-Ru Lai#

*Department of Plant Pathology and Microbiology, and #Department of Surgery, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan 106

 

Previously, we have characterized an intramolecular interaction within FAK protein, which plays a regulatory role in its activity. To further understand the molecular mechanism of this regulatory event, we compared FAKˇ¦s FERM-like domain with other FERM domains. In particular, several conserved residues which are important for other FERM domain containing proteinsˇ¦ regulation mediated by interaction with phosphoinositide were recognized and rendered for mutational analysis. We found that FAKˇ¦s FERM domain possesses a high affinity with D-3 and D-4 phosphoinositides and mutation on some of these conserved residues reduced the tyrosine phosphorylation and phospho-Tyr397 of FAK. These findings were consistent with the decreased tyrosine phosphorylation and kinase activity of FAK while the depletion of phosphate supply or blockage of phosphoinositides by presence of neomycin in the cell culture condition. Furthermore, we also found in the absence of phosphoinositide binding led to a significant decrease in cell motility whereas overexpression of wild type FAK or an activated form of FAK (N16-FAK) could rescue this phenotype. Interestingly, a plasma membrane targeting FAK exhibited a hyper-phosphorylated status and which was independent of the focal contact localization. Further experiments will focus on how the signaling events of FAK to mediate distinct cellular functions regulated by phosphoinositides. These studies will provide an insight on a complete picture of FAK activation in a molecular detail.