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Title: A hetero-micro-seeding strategy for readily crystallizing closely related protein variants

Abstract

Protein crystallization remains difficult to rationalize and screening for optimal crystallization conditions is a tedious and time consuming procedure. Here, we report a hetero-micro-seeding strategy for producing high resolution crystals of closely related protein variants, where micro crystals from a readily crystallized variant are used as seeds to develop crystals of other variants less amenable to crystallization. We applied this strategy to Bovine Pancreatic Trypsin Inhibitor (BPTI) variants, which would not crystallize using standard crystallization practice. Out of six variants in our analysis, only one called BPTI-[5,55]A14G formed well behaving crystals; and the remaining five (A14GA38G, A14GA38V, A14GA38L, A14GA38I, and A14GA38K) could be crystallized only using micro-seeds from the BPTI-[5,55]A14G crystal. All hetero-seeded crystals diffracted at high resolution with minimum mosaicity, retaining the same space group and cell dimension. Moreover, hetero-micro-seeding did not introduce any biases into the mutant’s structure toward the seed structure, as demonstrated by A14GA38I structures solved using micro-seeds from A14GA38G, A14GA38L and A14GA38I. Though hetero-micro-seeding is a simple and almost naïve strategy, this is the first direct demonstration of its workability. We believe that hetero-micro-seeding, which is contrasting with the popular idea that crystallization requires highly purified proteins, could contribute a new tool for rapidly solvingmore » protein structures in mutational analysis studies. - Highlights: • In hetero-micro-seeding, hard-to-crystalize mutants are crystallized using micro-seeds from a readily crystalized variant. • Hetero-micro-seeding did not introduce any biases of the mutant structure toward the seed structure. • Though hetero-micro-seeding is a simple strategy, this is the first direct demonstration of its workability at atomic level.« less

Authors:
 [1];  [2];  [1]
  1. Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588 (Japan)
  2. (Bangladesh)
Publication Date:
OSTI Identifier:
22719127
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 493; Journal Issue: 1; Other Information: Copyright (c) 2017 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; 60 APPLIED LIFE SCIENCES; CATTLE; CRYSTALLIZATION; CRYSTALS; DISULFIDES; LATTICE PARAMETERS; MUTANTS; PANCREAS; PROTEIN ENGINEERING; PROTEIN STRUCTURE; TRYPSIN

Citation Formats

Islam, Mohammad M., Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, 4331, and Kuroda, Yutaka. A hetero-micro-seeding strategy for readily crystallizing closely related protein variants. United States: N. p., 2017. Web. doi:10.1016/J.BBRC.2017.08.161.
Islam, Mohammad M., Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, 4331, & Kuroda, Yutaka. A hetero-micro-seeding strategy for readily crystallizing closely related protein variants. United States. doi:10.1016/J.BBRC.2017.08.161.
Islam, Mohammad M., Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, 4331, and Kuroda, Yutaka. Sat . "A hetero-micro-seeding strategy for readily crystallizing closely related protein variants". United States. doi:10.1016/J.BBRC.2017.08.161.
@article{osti_22719127,
title = {A hetero-micro-seeding strategy for readily crystallizing closely related protein variants},
author = {Islam, Mohammad M. and Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, 4331 and Kuroda, Yutaka},
abstractNote = {Protein crystallization remains difficult to rationalize and screening for optimal crystallization conditions is a tedious and time consuming procedure. Here, we report a hetero-micro-seeding strategy for producing high resolution crystals of closely related protein variants, where micro crystals from a readily crystallized variant are used as seeds to develop crystals of other variants less amenable to crystallization. We applied this strategy to Bovine Pancreatic Trypsin Inhibitor (BPTI) variants, which would not crystallize using standard crystallization practice. Out of six variants in our analysis, only one called BPTI-[5,55]A14G formed well behaving crystals; and the remaining five (A14GA38G, A14GA38V, A14GA38L, A14GA38I, and A14GA38K) could be crystallized only using micro-seeds from the BPTI-[5,55]A14G crystal. All hetero-seeded crystals diffracted at high resolution with minimum mosaicity, retaining the same space group and cell dimension. Moreover, hetero-micro-seeding did not introduce any biases into the mutant’s structure toward the seed structure, as demonstrated by A14GA38I structures solved using micro-seeds from A14GA38G, A14GA38L and A14GA38I. Though hetero-micro-seeding is a simple and almost naïve strategy, this is the first direct demonstration of its workability. We believe that hetero-micro-seeding, which is contrasting with the popular idea that crystallization requires highly purified proteins, could contribute a new tool for rapidly solving protein structures in mutational analysis studies. - Highlights: • In hetero-micro-seeding, hard-to-crystalize mutants are crystallized using micro-seeds from a readily crystalized variant. • Hetero-micro-seeding did not introduce any biases of the mutant structure toward the seed structure. • Though hetero-micro-seeding is a simple strategy, this is the first direct demonstration of its workability at atomic level.},
doi = {10.1016/J.BBRC.2017.08.161},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 1,
volume = 493,
place = {United States},
year = {2017},
month = {11}
}