Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Vilius Kurauskas,Paul Schanda,Jacques-Philippe Colletier,Nicolas Coquelle,Audrey Hessel,Tairan Yuwen,Sergei A Izmailov,Yi Xue,Nikolai R Skrynnikov,Isabel Ayala,Anastasya Shilova,Joyce Woodhouse,Olga N Rogacheva

doi:10.1038/s41467-017-00165-8

Abstract

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, near-rotary-resonance relaxation dispersion (NERRD) experiments probe angular backbone motion, while Bloch–McConnell relaxation dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with an ~3–5° amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.

Highlights

Proteins perform their functions in solution but their structures are most frequently studied inside crystals
Comparative molecular dynamics (MD) simulations of proteins embedded in an explicit crystal lattice and in solution produced useful insights, at least over the time scales accessible to MD22–25
The picture arising from these NMR and MD studies suggests that the crystal lattice has only little impact on dynamics on sub-microsecond time scales: changes are primarily found in loops engaged in crystal contacts, whereas secondary structure elements and loops remote from neighboring molecules showed similar order parameters

Summary

Introduction

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Near-rotary-resonance relaxation dispersion (NERRD) experiments probe angular backbone motion, while Bloch–McConnell relaxation dispersion data report on fluctuations of the local electronic environment These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. We use a combination of ssNMR experimental methods with microsecond-long MD simulations of explicit crystal lattices, in order to investigate how the crystalline environment impacts slow dynamics of the 8.6 kDa regulatory protein ubiquitin. The dynamics of this protein in solution have been extensively characterized experimentally and computationally. Our previous study did not provide a precise estimate of the rocking motion time scale, and only a range from ~100 ns to 100 μs was indicated[35]

Methods

Results

Conclusion