| Co-Investigator(Kenkyū-buntansha) |
SAZAKI Gen Inst for Materials Research, Tohoku University, Research Assoc., 金属材料研究所, 助手 (60261509)
MIYASHITA Satoru Inst for Materials Research, Tohoku University, Research Assoc., 金属材料研究所, 助手 (00219776)
吉川 彰 東北大学, 金属材料研究所, 助手 (50292264)
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| Budget Amount *help |
¥27,000,000 (Direct Cost: ¥27,000,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1998: ¥24,500,000 (Direct Cost: ¥24,500,000)
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| Research Abstract |
The goal of this work has been to extend the investigation of protein crystallization mechanisms to the level of individual molecules. Within this area, the research followed several directions : 1) use of atomic force microscopy (AFM) to follow the kinetics of protein crystal growth in situ, relating the observable parameters such as step velocity to molecular scale quantities such as bond energies ; 2) development of a modification of the conventional AFM technique to directly monitor single-molecule events during crystal growth, such as surface diffusion and incorporation ; 3) use of optical techniques to monitor properties which might be related to molecular level phenomena, such as alignment effects due to extremely high magnetic field, or changes in molecular hydration under high pressure. In addition, other research on crystal growth was carried out using the AFM and therefore considered to be indirectly supported. Regarding 1), AFM observation was used to elucidate the mechanis
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m of impurity action in inhibiting crystal growth of tetragonal form lysozyme, which can be understood in terms of a step-blocking model. AFM was also used to investigate the kinetics of step motion on monoclinic lysozyme crystals, which could be related to the intermolecular "macrobonds" which determined the morphology of the crystal. In connection with 2), we developed a modified commercial atomic force microscope combined with a high-speed data acquisition system to pursue more direct single-molecule measurements. The AFM, operated in tapping mode without scanning, gave a signal representing sample height changes due to motion of the topmost protein molecule under the tip. A much improved signal-to-noise ratio can be obtained by using very small cantilevers ; with these, the system should be capable of the ultimate goal of single-molecule observation. As for area 3), fluorescence microscopy observations were used to demonstrate the role of molecular charge and crystal growth rate on impurity incorporation, and polarization rotation and in situ optical microscopy were used to study magnetic field effects, which could be understood via the observed reduction of solution convection, but had at most small influence on molecular-scale parameters such as alignment. Meanwhile, understanding of the action of ever-present impurities and means of optimizing crystal morphology through solution conditions and external fields have already enabled the production of higher quality crystals for structure determination at higher resolution Less
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