Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal

X-ray crystallography is a major tool to provide three dimensional structures. High resolution protein crystal data is important in revealing a highly accurate structure at atomic level. This is essential in order to understand the properties and function of the protein. In crystallization process,...

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Main Author:	Mohamad Aris, Sayangku Nor Ariati
Format:	Thesis
Language:	English
Published:	2013
Subjects:	Crystallography Crystals Crystallization
Online Access:	http://psasir.upm.edu.my/id/eprint/39914/7/FBSB%202013%2017R.pdf
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spelling	my-upm-ir.399142019-11-29T08:35:46Z Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal 2013-05 Mohamad Aris, Sayangku Nor Ariati X-ray crystallography is a major tool to provide three dimensional structures. High resolution protein crystal data is important in revealing a highly accurate structure at atomic level. This is essential in order to understand the properties and function of the protein. In crystallization process, convection and sedimentation is thought to be detrimental for growth of high quality protein crystals. Both aspects can be avoided by either working in gelled systems, working in systems of small dimensions, or in the absence of gravity. In this research, crystallization by using counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Recombinant T1 Lipase from Geobacillus zalihae was successfully purified to homogeneity via two step affinity chromatography followed by ion exchange chromatography with 8.9 % yield. Purified T1 lipase was then crystallized in microbatch and vapour diffusion methods. The best crystal growth was obtained in Formulation 21 (Crystal Screen 2) with optimum growth temperature of 20 °C. The crystallization set up condition was applied to counter diffusion method for space experiment as well as ground control. Crystallization of T1 lipase under microgravity condition experiment was done in collaboration with JAXA (Japanese Aerospace Exploration Agency) under the JAXA-UPM Protein Crystal Growth (PCG) #2 Flight 2 program. The synchrotron diffraction data set were collected to 1.3 Å and 1.1 Å resolutions and belonged to monoclinic C2 Space group for ground grown and space grown crystal, respectively. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase. The structural analysis of T1 lipase was performed using molecular replacement method with final R factor 0.134 (Rfree 0.162) for ground-grown crystal and 0.129 (Rfree 0.150) for space-grown crystal,respectively. The structure of T1 lipase crystal contains two molecules per asymmetric unit with 387 amino acids each. Overall topology showed of α/β hydrolase fold containing catalytic triad active site and covered by α-helix 6 and α-helix 7 as a ‘lid’. The structure contained zinc and calcium binding site which was important for the structural stabilization. Comparative crystallographic analysis revealed that the space-grown crystal structure has improved as compared to ground grown crystal. This study has shown that crystallization in counter diffusion method using microgravity environment improved the internal order of crystals thus gave a more precise three dimensional structure. Crystallography Crystals Crystallization 2013-05 Thesis http://psasir.upm.edu.my/id/eprint/39914/ http://psasir.upm.edu.my/id/eprint/39914/7/FBSB%202013%2017R.pdf application/pdf en public masters Universiti Putra Malaysia Crystallography Crystals Crystallization
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Crystallography Crystals Crystallization
spellingShingle	Crystallography Crystals Crystallization Mohamad Aris, Sayangku Nor Ariati Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal
description	X-ray crystallography is a major tool to provide three dimensional structures. High resolution protein crystal data is important in revealing a highly accurate structure at atomic level. This is essential in order to understand the properties and function of the protein. In crystallization process, convection and sedimentation is thought to be detrimental for growth of high quality protein crystals. Both aspects can be avoided by either working in gelled systems, working in systems of small dimensions, or in the absence of gravity. In this research, crystallization by using counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Recombinant T1 Lipase from Geobacillus zalihae was successfully purified to homogeneity via two step affinity chromatography followed by ion exchange chromatography with 8.9 % yield. Purified T1 lipase was then crystallized in microbatch and vapour diffusion methods. The best crystal growth was obtained in Formulation 21 (Crystal Screen 2) with optimum growth temperature of 20 °C. The crystallization set up condition was applied to counter diffusion method for space experiment as well as ground control. Crystallization of T1 lipase under microgravity condition experiment was done in collaboration with JAXA (Japanese Aerospace Exploration Agency) under the JAXA-UPM Protein Crystal Growth (PCG) #2 Flight 2 program. The synchrotron diffraction data set were collected to 1.3 Å and 1.1 Å resolutions and belonged to monoclinic C2 Space group for ground grown and space grown crystal, respectively. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase. The structural analysis of T1 lipase was performed using molecular replacement method with final R factor 0.134 (Rfree 0.162) for ground-grown crystal and 0.129 (Rfree 0.150) for space-grown crystal,respectively. The structure of T1 lipase crystal contains two molecules per asymmetric unit with 387 amino acids each. Overall topology showed of α/β hydrolase fold containing catalytic triad active site and covered by α-helix 6 and α-helix 7 as a ‘lid’. The structure contained zinc and calcium binding site which was important for the structural stabilization. Comparative crystallographic analysis revealed that the space-grown crystal structure has improved as compared to ground grown crystal. This study has shown that crystallization in counter diffusion method using microgravity environment improved the internal order of crystals thus gave a more precise three dimensional structure.
format	Thesis
qualification_level	Master's degree
author	Mohamad Aris, Sayangku Nor Ariati
author_facet	Mohamad Aris, Sayangku Nor Ariati
author_sort	Mohamad Aris, Sayangku Nor Ariati
title	Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal
title_short	Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal
title_full	Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal
title_fullStr	Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal
title_full_unstemmed	Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal
title_sort	crystallographic analysis of ground- and space-grown thermostable t1 lipase crystal
granting_institution	Universiti Putra Malaysia
publishDate	2013
url	http://psasir.upm.edu.my/id/eprint/39914/7/FBSB%202013%2017R.pdf
_version_	1747811826003869696

Crystallographic analysis of ground- and space-grown thermostable T1 lipase crystal

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