Size and Shape Controlled Crystallization of Hemoglobin for Advanced Crystallography

Ayana Sato-Tomita,Naoya Shibayama

doi:10.3390/cryst7090282

Abstract

While high-throughput screening for protein crystallization conditions have rapidly evolved in the last few decades, it is also becoming increasingly necessary for the control of crystal size and shape as increasing diversity of protein crystallographic experiments. For example, X-ray crystallography (XRC) combined with photoexcitation and/or spectrophotometry requires optically thin but well diffracting crystals. By contrast, large-volume crystals are needed for weak signal experiments, such as neutron crystallography (NC) or recently developed X-ray fluorescent holography (XFH). In this article, we present, using hemoglobin as an example protein, some techniques for obtaining the crystals of controlled size, shape, and adequate quality. Furthermore, we describe a few case studies of applications of the optimized hemoglobin crystals for implementing the above mentioned crystallographic experiments, providing some hints and tips for the further progress of advanced protein crystallography.

Highlights

Since the landmark work by Perutz and Kendrew on the X-ray structural determination of hemoglobin (Hb) and myoglobin (Mb) in the 1950s [1,2,3], X-ray crystallography (XRC) has been the most powerful technique to determine protein structures at the atomic level
1980s, with the advances in highly intense synchrotron X-ray sources, computer power, experimental instruments, recombinant protein expression systems, phase determination techniques, and high-throughput/rationally-designed screening for crystallization conditions. Along with these technological and methodological advances, the crystal size and time required for XRC have been steadily decreasing over the years, and the initial search of crystallization conditions has become much easier and less time consuming
Even though XRC has provided atomic details of numerous protein structures, a mechanistic description of proteins requires information about intermediates that occur during protein functioning

Summary

Introduction

Since the landmark work by Perutz and Kendrew on the X-ray structural determination of hemoglobin (Hb) and myoglobin (Mb) in the 1950s [1,2,3], X-ray crystallography (XRC) has been the most powerful technique to determine protein structures at the atomic level. The identification of protein crystallization conditions was usually a trial-and-error process relied largely on the empirical knowledge of individual researchers This is well illustrated by the fact that only eleven protein structures were solved by 1970. 1980s, with the advances in highly intense synchrotron X-ray sources, computer power, experimental instruments (such as X-ray detectors and automated sample manipulators), recombinant protein expression systems, phase determination techniques, and high-throughput/rationally-designed screening for crystallization conditions. Along with these technological and methodological advances, the crystal size and time required for XRC have been steadily decreasing over the years, and the initial search of crystallization conditions has become much easier and less time consuming. The Protein Data Bank (PDB) [4] contains more than 120,000 protein structures, with the majority of them determined by XRC

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