Tracing transport of protein aggregates in microgravity versus unit gravity crystallization

Arayik Martirosyan,Martin Cox,Jan Knop,Lawrence J Delucas,Christian Betzel,Deborah Mccombs,Sven Falke,Christopher D Radka

doi:10.1038/s41526-022-00191-x

Abstract

Microgravity conditions have been used to improve protein crystallization from the early 1980s using advanced crystallization apparatuses and methods. Early microgravity crystallization experiments confirmed that minimal convection and a sedimentation-free environment is beneficial for growth of crystals with higher internal order and in some cases, larger volume. It was however realized that crystal growth in microgravity requires additional time due to slower growth rates. The progress in space research via the International Space Station (ISS) provides a laboratory-like environment to perform convection-free crystallization experiments for an extended time. To obtain detailed insights in macromolecular transport phenomena under microgravity and the assumed reduction of unfavorable impurity incorporation in growing crystals, microgravity and unit gravity control experiments for three different proteins were designed. To determine the quantity of impurity incorporated into crystals, fluorescence-tagged aggregates of the proteins (acting as impurities) were prepared. The recorded fluorescence intensities of the respective crystals reveal reduction in the incorporation of aggregates under microgravity for different aggregate quantities. The experiments and data obtained, provide insights about macromolecular transport in relation to molecular weight of the target proteins, as well as information about associated diffusion behavior and crystal lattice formation. Results suggest one explanation why microgravity-grown protein crystals often exhibit higher quality. Furthermore, results from these experiments can be used to predict which proteins may benefit more from microgravity crystallization.

Highlights

Proteins are vital and important macromolecules without which our bodies and other living organisms would be unable to repair, regulate, or protect against unwanted infectious organisms
It was found that the average growth rate of the major axis of needle-shaped Plasmodium falciparum glutathione-Stransferase (PfGST) crystals and lysozyme crystals was found to be higher (p < 0.01) at unit gravity compared to the microgravity environment
Results from comparative crystal growth investigations showed a clear decrease in crystal growth rates along the major axis for microgravity-grown crystals of lysozyme and an increase in the length of the major axis for PfGST crystals

Summary

Introduction

Proteins (we will use the term protein consistently to cover other bio-macromolecules such as nucleic acids and proteinnucleic acid complexes as well) are vital and important macromolecules without which our bodies and other living organisms would be unable to repair, regulate, or protect against unwanted infectious organisms. X-ray crystallography is the most efficient method to determine protein structures, the technique requires growth of protein crystals of sufficient quality. Protein crystals grown in microgravity, initially utilizing unmanned rockets and subsequently on US space shuttle missions[1], resulted in clear crystal quality improvements, reported for several investigations via X-ray diffraction analysis[2–10]. Past microgravity experiments have investigated two possible reasons for the improved crystal quality that is often observed. This investigation provides experimental data to address the two prevailing theories regarding why a microgravity environment sometimes yields protein crystals of superior quality. The following hypothesis was addressed: Improved quality of microgravity-grown protein crystals is the result of two macromolecular characteristics that exist in a buoyancy-free, diffusion-dominated solution:

Methods

Results

Conclusion