Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

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.

Internal protein motions in molecular-dynamics simulations of Bragg and diffuse X-ray scattering

“…Higher quality data also might be needed to substantially improve the model. Recent solid-state NMR (ssNMR) experiments combined with crystalline protein simulations (Kurauskas et al, 2017;Ma et al, 2015;Mollica et al, 2012) create opportunities for joint validation of MD simulations using crystallography and NMR. An ssNMR + MD study of the protein GB1 (Mollica et al, 2012) showed reasonable agreement between 200 ns MD simulations and the data for longitudinal relaxation rates and chemical shifts, with lower agreement for the transverse relaxation rates.…”

“…An ssNMR + MD study of the protein GB1 (Mollica et al, 2012) showed reasonable agreement between 200 ns MD simulations and the data for longitudinal relaxation rates and chemical shifts, with lower agreement for the transverse relaxation rates. An ssNMR + MD study of ubiquitin (Kurauskas et al, 2017;Ma et al, 2015) attributed the transverse relaxation rates to rigid-body rotations of whole proteins in the crystal lattice, with amplitudes of 3-5 extracted from the simulations. To assess the importance of rigid-body rotations in the present staphylococcal nuclease simulation, snapshots of each of the 32 copies of the protein were rotationally aligned with a reference structure (x2).…”

Microsecond Protein Dynamics from Combined Bloch‐McConnell and Near‐Rotary‐Resonance R1p Relaxation‐Dispersion MAS NMR

“…Ubiquitin is a convenient model system, because its μ s dynamics has been characterized extensively by solution‐NMR, solid‐state NMR and MD simulations ,,. The exchange process involves a flip of the peptide plane of D52/G53, along with reorientation of the side chain of E24, which is H‐bonded in one of these two states, but not in the other state.…”

“…As an important consequence of this dependence on the spectral density function at sums and differences of and , in the presence of μ s motion, an increase of R 1ρ is observed as the RF field strength and the MAS frequency become similar. This effect, described by Redfield theory, and shown in several studies,, was dubbed “NEar‐Rotary Resonance RelaxationDispersion” (NERRD) recently . We use this term throughout this manuscript.…”

Because the Light is Better Here: Correlation‐Time Analysis by NMR Spectroscopy

“…We did so for several dynamics studies and compared the result to the reported τ c values. The biasing/reported τ c for a number of studies are: HET‐s(218–289) 15 N 17 μs/19 μs, HET‐s(218–289) 13 C α 11 μs/7 μs, ubiquitin (2‐methyl‐2,4‐pentanediol (MPD) crystallization) 2.1 μs/1.5 μs (median value), ubiquitin (PEG crystallization) 26 μs/20 μs (SI, Figure 5) . The good agreement between these numbers suggests that the real motion is too complex to be described with a mono‐exponential correlation function, so that results bias to where the experiments are sensitive.…”