William W. Brey

M2 Proton Channel Structural Validation from Full Length Protein Samples in Synthetic Bilayers and E. coli Membranes

Membrane protein structure and function, especially for small membrane proteins, can be highly sensitive to the membrane mimetic environment used for structural characterization, as exemplified by the M2 protein from influenza A virus that has been characterized in liquid crystalline lipid bilayers, detergent micelles and in detergent based crystals.[3–8] Various transmembrane (TM) helical tilt angles, different drug binding sites and amphipathic helix interactions, as well as a lack of consensus on the sidechain geometry for the functionally critical residues is apparent from this set of structures. Many of these structural differences can be explained based on the influence of the proteins environment. Hydrophobic thickness influences the helical tilt; detergent penetration into the helical bundle and crystal contacts influence the packing and hence tilt of the helices, while the highly curved surface of micelles destabilize the interactions of amphipathic helices with what would be the bilayer interface.[9] These structural perturbations can influence functional properties such as the binding of the antiviral drug to the protein and our understanding of the proton channel functional mechanism. Exactly how well the native membrane needs to be modeled to achieve a native membrane protein structure is explored here, where we aim to validate the structure of the tetrameric M2 conductance domain (M2CD; residues 22–62; PDB #2L0J) that has been structurally characterized in synthetic lipid bilayers. We have set out to do this by observing the full length protein in synthetic bilayers, as well as in native E. coli membranes. For the first time we report on structural insights from the full length M2 (M2FL) protein using magic angle spinning solid state NMR (ssNMR) and we present spectra of the protein as it is inserted into the E. coli membranes by the cellular apparatus without ever being exposed to a detergent environment. These results validate the earlier structural results obtained from the M2CD observed in a liquid crystalline bilayer envionment.

Magic Angle Spinning NMR Reveals Sequence-Dependent Structural Plasticity, Dynamics, and the Spacer Peptide 1 Conformation in HIV-1 Capsid Protein Assemblies

A key stage in HIV-1 maturation toward an infectious virion requires sequential proteolytic cleavage of the Gag polyprotein leading to the formation of a conical capsid core that encloses the viral RNA genome and a small complement of proteins. The final step of this process involves severing the SP1 peptide from the CA-SP1 maturation intermediate, which triggers the condensation of the CA protein into the capsid shell. The details of the overall mechanism, including the conformation of the SP1 peptide in CA-SP1, are still under intense debate. In this report, we examine tubular assemblies of CA and the CA-SP1 maturation intermediate using magic angle spinning (MAS) NMR spectroscopy. At magnetic fields of 19.9 T and above, outstanding quality 2D and 3D MAS NMR spectra were obtained for tubular CA and CA-SP1 assemblies, permitting resonance assignments for subsequent detailed structural characterization. Dipolar- and scalar-based correlation experiments unequivocally indicate that SP1 peptide is in a random coil conformation and mobile in the assembled CA-SP1. Analysis of two CA protein sequence variants reveals that, unexpectedly, the conformations of the SP1 tail, the functionally important CypA loop, and the loop preceding helix 8 are modulated by residue variations at distal sites. These findings provide support for the role of SP1 as a trigger of the disassembly of the immature CA capsid for its subsequent de novo reassembly into mature cores and establish the importance of sequence-dependent conformational plasticity in CA assembly.

Nuclear magnetic resonance probe coil with enhanced current-carrying capability

The conductive material in an RF coil disposed in the polarizing field of an NMR apparatus in miminized and the current density at each point in the coil kept constant by providing an inductive element and a set of tapered, interidigtated capacitors having a uniform gap therebetween. The invention maximizes the current-carrying capacity of the coil.

Dynamic allostery governs cyclophilin A-HIV capsid interplay

Significance The mechanisms of how Cyclophilin A (CypA) regulates HIV-1 infectivity remain poorly understood. We examined the role of dynamics in capsid (CA) protein assemblies by magic-angle-spinning NMR. The assembled CA is highly dynamic. Dipolar tensors calculated from molecular dynamics trajectories are in quantitative agreement with the NMR results. Motions in the CypA loop are sequence-dependent and attenuated in the escape mutants A92E and G94D. Dynamics are similar in escape mutants and CA/CypA complex. These findings suggest that CA escapes from CypA dependence through dynamic allostery. Thus, a host factors function in HIV infectivity may not be primarily associated with a structural change of the capsid core, but with altering its dynamics, such as the reduction of motions for the CypA loop. Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone 1H-15N and 1H-13C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1s escape from CypA dependence.

Magic angle spinning and oriented sample solid-state NMR structural restraints combine for influenza a M2 protein functional insights.

As a small tetrameric helical membrane protein, the M2 proton channel structure is highly sensitive to its environment. As a result, structural data from a lipid bilayer environment have proven to be essential for describing the conductance mechanism. While oriented sample solid-state NMR has provided a high-resolution backbone structure in lipid bilayers, quaternary packing of the helices and many of the side-chain conformations have been poorly restrained. Furthermore, the quaternary structural stability has remained a mystery. Here, the isotropic chemical shift data and interhelical cross peaks from magic angle spinning solid-state NMR of a liposomal preparation strongly support the quaternary structure of the transmembrane helical bundle as a dimer-of-dimers structure. The data also explain how the tetrameric stability is enhanced once two charges are absorbed by the His37 tetrad prior to activation of this proton channel. The combination of these two solid-state NMR techniques appears to be a powerful approach for characterizing helical membrane protein structure.

Initial in vivo rodent sodium and proton MR imaging at 21.1 T

The first in vivo sodium and proton magnetic resonance (MR) images and localized spectra of rodents were attained using the wide bore (105 mm) high resolution 21.1-T magnet, built and operated at the National High Magnetic Field Laboratory (Tallahassee, FL, USA). Head images of normal mice (C57BL/6J) and Fisher rats (approximately 250 g) were acquired with custom designed radiofrequency probes at frequencies of 237/900 MHz for sodium and proton, respectively. Sodium MR imaging resolutions of approximately 0.125 microl for mouse and rat heads were achieved by using a 3D back-projection pulse sequence. A gain in SNR of approximately 3 for sodium and approximately 2 times for proton were found relative to corresponding MR images acquired at 9.4 T. 3D Fast Low Angle Shot (FLASH) proton mouse images (50x50x50 microm(3)) were acquired in 90 min and corresponding rat images (100x100x100 microm(3)) within a total time of 120 min. Both in vivo large rodent MR imaging and localized spectroscopy at the extremely high field of 21.1 T are feasible and demonstrate improved resolution and sensitivity valuable for structural and functional brain analysis.

High-field NMR using resistive and hybrid magnets.

Resistive and resistive-superconducting hybrid magnets can generate dc magnetic fields much higher than conventional superconducting NMR magnets but the field spatial homogeneity and temporal stability are usually not sufficient for high-resolution NMR experiments. Hardware and technique development addressing these issues are presented for high-resolution NMR at magnetic fields up to 40T. Passive ferromagnetic shimming and magic-angle spinning are used effectively to reduce the broadening from inhomogeneous magnetic field. A phase correction technique based on simultaneous heteronuclear detection is developed to compensate magnetic field fluctuations to achieve high spectral resolution.

Development of a 13C-optimized 1.5-mm high temperature superconducting NMR probe

We report a 1.5-mm NMR probe based on high temperature superconductors operating at 14.1T optimized for (13)C detection. The probe has a total sample volume of about 35 microliters (μL) with an active volume of 20 μL and provides exceptional mass sensitivity for (13)C detection. The probe also has excellent (1)H sensitivity and employs a (2)H lock; (15)N irradiation capability can be added in the future. The coils are cooled to about 20K using a standard Agilent cryogenic refrigeration system, and the sample temperature is regulated near room temperature. The coil design considerations are discussed in detail. This probe is ideal for directly detected (13)C NMR experiments for natural products chemistry and metabolomics applications, for which 35 μL is an optimal sample volume. The outstanding (13)C sensitivity of this probe allowed us to directly determine the (13)C connectivity on 1.1mg of natural abundance histidine using an INADEQUATE experiment. We demonstrated the utility of this probe for (13)C-based metabolomics using a synthetic mixture of common natural abundance metabolites whose concentrations ranged from 1 to 5mM (40-200 nmol).

Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers.

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1H-15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([1H,15N]-SE-PISEMA-PDSD). The incorporation of 2D 15N/15N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.

Enhancing MQMAS of low-γ nuclei by using a high B1 field balanced probe circuit

A balanced probe circuit is used to generate high B(1) magnetic field for sensitivity enhancement of multiple-quantum magic-angle spinning (MQMAS) experiment applied to low-gamma quadrupolar nuclei. Electrical balancing of the sample coil can cut the peak voltage by a half, therefore improving the power handling when generating a two-fold higher B(1) field. Experimental results, illustrated here with (25)Mg data for two layered double hydroxides, show that the MQMAS efficiency increases more than linearly with the B(1) field strength. The multiplicative enhancements from high B(0) and B(1) fields and an optimized MQMAS pulse sequence provide the critically needed sensitivity for acquiring MQMAS spectra of low-gamma quadrupolar nuclei such as (25)Mg at natural abundance.