10:45 AM - 11:15 AM
[MIS13-06] Effects of microgravity on lateral velocities of the spiral growth hillocks of protein crystals
★Invited Papers
Keywords:microgravity, protein crystal, glucose isomerase, in situ observation, two beam interferometer
High-quality protein crystals with their maximum resolution of diffraction higher than 1.5 Å are pre-requisites for structure- based drug design. Thus, methods for crystallizing high- quality protein crystals have been required.
NASA reported that about 20% of the protein crystals grown in space exhibit higher X-ray resolution limits than those of the best crystals grown on the earth. Although many studies on protein crystallization in space were conducted for obtaining such high-quality protein crystals, the exact mechanisms leading to the improvement in the quality under microgravity conditions have not been sufficiently studied due to the scarcity of highly purified protein samples.
Accurate measurements of the growth of molecular steps (step velocity Vstep) on crystals in supersaturated solution using a sufficient quantity of highly purified protein would enable us to clarify the effects of gravity on the elementary processes of crystal growth directly.
In 2012, Tsukamoto conducted the NanoStep mission on the Japanese Experiment Module (KIBO) of the International Space Station (ISS) and measured lateral velocity Vlateral in the <110> direction of spiral growth hillocks on the {110} face of a highly purified (99.8% purity) tetragonal hen egg-white lysozyme (HEWL) crystal in situ under microgravity for the first time [1, 2]. The values of Vlateral were almost equal to (or even faster than) those of Vstep in the <110> direction of spiral growth hillocks measured on the earth. We also measured the values of Vlateral of highly purified (99.9% purity) glucose isomerase crystals under microgravity conditions in the Advanced Nano Step (AdNano) mission of ISS KIBO experiments [3]. They were almost equal to those of Vstep in the same direction measured on the earth. These results showed that the thorough purification would enable us to escape from the usage of the ISS for obtaining high quality protein crystals.
NanoStep mission also revealed that the values of Vlateral of lysozyme crystals in existence with covalently bound lysozyme dimers as large impurities under microgravity conditions was faster than those on the earth.2 The effective segregation coefficient of the lysozyme dimer which is larger than unity would be the main reason. In this case, microgravity conditions are favorable for improvements of crystal quality.
In contrast, the values of Vlateral of glucose isomerase crystals in existence with hemoglobin as an small impurity measured under microgravity conditions in the AdNano mission were slower than those those of Vstep in the same direction measured on the earth. In this case, microgravity conditions are unfavorable; space experiments are not always useful to improve the quality of protein crystals.
This study was supported by JAXA. The authors also thank Professor Alexander Chernov of Lawrence Livermore National Laboratory for valuable suggestions and discussion. Y.S. was partially supported by JSPS KAKENHI Grant No. 24656016, 26390054 , 15K05668, 16K05470, 18K04960, 20K03782, and 21K04908. K.T. was supported by JSPS KAKENHI Grant No. 22244066. The authors sincerely thank Kanae Yagi of the Center for Administration of Information Technology, Tokushima University for her accurate technical support of live feed of microscopic images from the ISS to Tokushima University. The authors thank Prof. Gen Sazaki of Hokkaido University, Prof. Masahide Sato and Professor Hiroyasu Katsuno of Kanazawa University for valuable discussion in the Joint Research Program of the Institute of Low Temperature Science, Hokkaido University.
[1] Yoshizaki, I.; Tsukamoto, K.; Yamazaki, T.; Murayama, K.; Oshi, K.; Fukuyama, S.; Shimaoka, T.; Suzuki, Y.; Tachibana, M. Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry. Rev. Sci. Instrum. 2013, 84, 103707.
[2] Suzuki, Y.; Tsukamoto, K.; Yoshizaki, I.; Miura, H.; Fujiwara, T. First direct observation of impurity effects on the growth rate of tetragonal lysozyme crystals under microgravity as measured by interferometry. Cryst. Growth Des. 2015, 15, 4787−4794.
[3] Suzuki, Y.; Ninomiya, A.; Fukuyama, S.; Shimaoka, T.; Nagai, M.; Inaka, K.; Yanagiya, S.; Sone, T.; Wachi S.; Kawaguchi, S.; Arai, Y.; Tsukamoto, K. Highly Purified Glucose Isomerase Crystals under Microgravity Conditions Grow as Fast as Those on the Ground Do. Cryst. Growth Des. 2022, 22, 7074-7078.
NASA reported that about 20% of the protein crystals grown in space exhibit higher X-ray resolution limits than those of the best crystals grown on the earth. Although many studies on protein crystallization in space were conducted for obtaining such high-quality protein crystals, the exact mechanisms leading to the improvement in the quality under microgravity conditions have not been sufficiently studied due to the scarcity of highly purified protein samples.
Accurate measurements of the growth of molecular steps (step velocity Vstep) on crystals in supersaturated solution using a sufficient quantity of highly purified protein would enable us to clarify the effects of gravity on the elementary processes of crystal growth directly.
In 2012, Tsukamoto conducted the NanoStep mission on the Japanese Experiment Module (KIBO) of the International Space Station (ISS) and measured lateral velocity Vlateral in the <110> direction of spiral growth hillocks on the {110} face of a highly purified (99.8% purity) tetragonal hen egg-white lysozyme (HEWL) crystal in situ under microgravity for the first time [1, 2]. The values of Vlateral were almost equal to (or even faster than) those of Vstep in the <110> direction of spiral growth hillocks measured on the earth. We also measured the values of Vlateral of highly purified (99.9% purity) glucose isomerase crystals under microgravity conditions in the Advanced Nano Step (AdNano) mission of ISS KIBO experiments [3]. They were almost equal to those of Vstep in the same direction measured on the earth. These results showed that the thorough purification would enable us to escape from the usage of the ISS for obtaining high quality protein crystals.
NanoStep mission also revealed that the values of Vlateral of lysozyme crystals in existence with covalently bound lysozyme dimers as large impurities under microgravity conditions was faster than those on the earth.2 The effective segregation coefficient of the lysozyme dimer which is larger than unity would be the main reason. In this case, microgravity conditions are favorable for improvements of crystal quality.
In contrast, the values of Vlateral of glucose isomerase crystals in existence with hemoglobin as an small impurity measured under microgravity conditions in the AdNano mission were slower than those those of Vstep in the same direction measured on the earth. In this case, microgravity conditions are unfavorable; space experiments are not always useful to improve the quality of protein crystals.
This study was supported by JAXA. The authors also thank Professor Alexander Chernov of Lawrence Livermore National Laboratory for valuable suggestions and discussion. Y.S. was partially supported by JSPS KAKENHI Grant No. 24656016, 26390054 , 15K05668, 16K05470, 18K04960, 20K03782, and 21K04908. K.T. was supported by JSPS KAKENHI Grant No. 22244066. The authors sincerely thank Kanae Yagi of the Center for Administration of Information Technology, Tokushima University for her accurate technical support of live feed of microscopic images from the ISS to Tokushima University. The authors thank Prof. Gen Sazaki of Hokkaido University, Prof. Masahide Sato and Professor Hiroyasu Katsuno of Kanazawa University for valuable discussion in the Joint Research Program of the Institute of Low Temperature Science, Hokkaido University.
[1] Yoshizaki, I.; Tsukamoto, K.; Yamazaki, T.; Murayama, K.; Oshi, K.; Fukuyama, S.; Shimaoka, T.; Suzuki, Y.; Tachibana, M. Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry. Rev. Sci. Instrum. 2013, 84, 103707.
[2] Suzuki, Y.; Tsukamoto, K.; Yoshizaki, I.; Miura, H.; Fujiwara, T. First direct observation of impurity effects on the growth rate of tetragonal lysozyme crystals under microgravity as measured by interferometry. Cryst. Growth Des. 2015, 15, 4787−4794.
[3] Suzuki, Y.; Ninomiya, A.; Fukuyama, S.; Shimaoka, T.; Nagai, M.; Inaka, K.; Yanagiya, S.; Sone, T.; Wachi S.; Kawaguchi, S.; Arai, Y.; Tsukamoto, K. Highly Purified Glucose Isomerase Crystals under Microgravity Conditions Grow as Fast as Those on the Ground Do. Cryst. Growth Des. 2022, 22, 7074-7078.