Direct observation of hierarchical protein dynamics

One of the fundamental challenges of physical biology is to understand the relationship between protein dynamics and function. At physiological temperatures, functional motions arise from the complex interplay of thermal motions of proteins and their environments. Here, we determine the hierarchy in the protein conformational energy landscape that underlies these motions, based on a series of temperature-dependent magic-angle spinning multinuclear nuclear-magnetic-resonance relaxation measurements in a hydrated nanocrystalline protein. The results support strong coupling between protein and solvent dynamics above 160 kelvin, with fast solvent motions, slow protein side-chain motions, and fast protein backbone motions being activated consecutively. Low activation energy, small-amplitude local motions dominate at low temperatures, with larger-amplitude, anisotropic, and functionally relevant motions involving entire peptide units becoming dominant at temperatures above 220 kelvin

Slice & Dice : nested spin–lattice relaxation measurements

Spin–lattice relaxation rate (R1) measurements are commonly used to characterize protein dynamics. However, the time needed to collect the data can be quite long due to long relaxation times of the low-gamma nuclei, especially in the solid state. We present a method to collect backbone heavy atom relaxation data by nesting the collection of datasets in the solid state. This method results in a factor of 2 to 2.5 times faster data acquisition for backbone R1 relaxation data for the 13C and 15N sites of proteins

Modulation of transmembrane domain interactions in neu receptor tyrosine kinase by membrane fluidity and cholesterol

The activation mechanism of the ErbB family of receptors is of considerable medical interest as they are linked to a number of human cancers, including an aggressive form of breast cancer. In the rat analogue of the human ErbB2 receptor, referred to as Neu, a point mutation in the transmembrane domain (V664E) has been shown to trigger oncogenic transformation. While the structural impact of this mutation has been widely studied in the past to yield models for the active state of the Neu receptor, little is known about the impact of cholesterol on its structure. Given previous reports of the influence of cholesterol on other receptor tyrosine kinases (RTKs), as well as the modulation of lipid composition in cancer cells, we wished to investigate how cholesterol content impacts the structure of the Neu transmembrane domain. We utilized high-resolution magic angle spinning solid-state NMR to measure 13C–13C coupling of selectively labelled probe residues in the Neu transmembrane domain in lipid bilayers containing cholesterol. We observe inter-helical coupling between residues that support helix–helix interactions on both dimerization motifs reported in the literature (A661-XXX-G665 and I659-XXX-V663). We further explore how changes in cholesterol concentration alter transmembrane domain interactions and the properties and mechanics of the bilayer. We interpret our results in light of previous studies relating RTK activity to cholesterol enrichment and/or depletion, and propose a novel model to explain our data that includes the recognition and binding of cholesterol by the Neu transmembrane domain through a putative cholesterol-recognition/interaction amino acid consensus sequence

Unraveling the complexity of protein backbone dynamics with combined 13C and 15N solid-state NMR relaxation measurements

Typically, protein dynamics involve a complex hierarchy of motions occurring on different time scales between conformations separated by a range of different energy barriers. NMR relaxation can in principle provide a site-specific picture of both the time scales and amplitudes of these motions, but independent relaxation rates sensitive to fluctuations in different time scale ranges are required to obtain a faithful representation of the underlying dynamic complexity. This is especially pertinent for relaxation measurements in the solid state, which report on dynamics in a broader window of time scales by more than 3 orders of magnitudes compared to solution NMR relaxation. To aid in unraveling the intricacies of biomolecular dynamics we introduce 13C spin–lattice relaxation in the rotating frame (R1ρ) as a probe of backbone nanosecond-microsecond motions in proteins in the solid state. We present measurements of 13C′ R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz 1H Larmor frequencies and compare them to 13C′ R1, 15N R1 and R1ρ measured under the same conditions. The addition of carbon relaxation data to the model free analysis of nitrogen relaxation data leads to greatly improved characterization of time scales of protein backbone motions, minimizing the occurrence of fitting artifacts that may be present when 15N data is used alone. We also discuss how internal motions characterized by different time scales contribute to 15N and 13C relaxation rates in the solid state and solution state, leading to fundamental differences between them, as well as phenomena such as underestimation of picosecond-range motions in the solid state and nanosecond-range motions in solution

Accelerating 15N and 13C R1 and R1q relaxation measurements by multiple pathway solid-state NMR experiments

Magic angle spinning (MAS) Solid-state NMR is a powerful technique to probe dynamics of biological systems at atomic resolution. R1 and R1ρ relaxation measurements can provide detailed insight on amplitudes and time scales of motions, especially when information from several different site-specific types of probes is combined. However, such experiments are time-consuming to perform. Shortening the time necessary to record relaxation data for different nuclei will greatly enhance practicality of such approaches. Here, we present staggered acquisition experiments to acquire multiple relaxation experiments from a single excitation to reduce the overall experimental time. Our strategy enables one to collect 15N and 13C relaxation data in a single experiment in a fraction of the time necessary for two separate experiments, with the same signal to noise ratio

Simultaneous MQMAS NMR experiments for two half-integer quadrupolar nuclei

A procedure to acquire two Multiple-Quantum Magic Angle Spinning (MQMAS) NMR experiments with the same instrument time is presented. A triply tuned probe is utilized with multiple receivers to collect data with staggered acquisitions and thus more efficiently use the instrument time. The data for one nucleus is collected during the recovery delay of the other nucleus, and vice versa. The instrument time is reduced to 60-80% of the time needed for the single acquisition collection Specifically our approach is presented for recording triple-quantum (3Q) 17O and either 3Q or quintuple-quantum (5Q) 27Al MAS NMR spectra of a 1.18Na2O•5SiO2•Al2O3 glass gel

Dipolar order parameters in large systems with fast spinning

Order parameters are a useful tool for quantifying amplitudes of molecular motions. Here we measure dipolar order parameters by recoupling heteronuclear dipole-dipole couplings under fast spinning. We apply symmetry based recoupling methods to samples spinning under magic angle at 60 kHz by employing a variable flip angle compound inversion pulse. We validate the methods by measuring site-specific 15N-1H order parameters of a microcrystalline protein over a small temperature range and the same protein in a large, precipitated complex with antibody. The measurements of the order parameters in the complex are consistent with the observed protein undergoing overall motion within the assembly

Nuclear spin diffusion under fast magic-angle spinning in solid-state NMR

Solid-state nuclear spin diffusion is the coherent and reversible process through which spin order is transferred via dipolar couplings. With the recent increases in magic-angle spinning (MAS) frequencies and magnetic fields becoming routinely applied in solid-state nuclear magnetic resonance, understanding how the increased 1H resolution obtained affects spin diffusion is necessary for interpretation of several common experiments. To investigate the coherent contributions to spin diffusion with fast MAS, we have developed a low-order correlation in Liouville space model based on the work of Dumez et al. (J. Chem. Phys. 33, 224501, 2010). Specifically, we introduce a new method for basis set selection, which accounts for the resonance-offset dependence at fast MAS. Furthermore, we consider the necessity of including chemical shift, both isotropic and anisotropic, in the modeling of spin diffusion. Using this model, we explore how different experimental factors change the nature of spin diffusion. Then, we show case studies to exemplify the issues that arise in using spin diffusion techniques at fast spinning. We show that the efficiency of polarization transfer via spin diffusion occurring within a deuterated and 100% back-exchanged protein sample at 60 kHz MAS is almost entirely dependent on resonance offset. We additionally identify temperature-dependent magnetization transfer in beta-aspartyl L-alanine, which could be explained by the influence of an incoherent relaxation-based nuclear Overhauser effect

Isolation and structural characterisation of rhodium(III) η2–fluoroarene complexes : experimental verification of predicted regioselectivity

The isolation and solid-state characterisation of complexes featuring partially coordinated benzene, fluorobenzene and all three isomers of difluorobenzene are described. Supported by a DFT analysis, this well-defined homologous series demonstrates the preference for η2-coordination of fluoroarenes via the HC[double bond, length as m-dash]CH sites adjacent to a fluorine substituent

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s Disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intra- and inter-molecular contacts, backbone and side chain torsion angles, relaxation measurements, and calculations of chemical shifts. These reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are co-assembled from differently structured monomers, which we describe as a type of ‘intrinsic’ polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. Weshow that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain, and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms