Peter L. Gor’kov

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.

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.

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.

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.

A volume birdcage coil with an adjustable sliding tuner ring for neuroimaging in high field vertical magnets: Ex and in vivo applications at 21.1 T

A tunable 900 MHz transmit/receive volume coil was constructed for ¹H MR imaging of biological samples in a 21.1 T vertical bore magnet. To accommodate a diverse range of specimen and RF loads at such a high frequency, a sliding-ring adaptation of a low-pass birdcage was implemented through simultaneous alteration of distributed capacitance. To make efficient use of the constrained space inside the vertical bore, a modular probe design was implemented with a bottom-adjustable tuning and matching apparatus. The sliding ring coil displays good homogeneity and sufficient tuning range for different samples of various dimensions representing large span of RF loads. High resolution in vivo and ex vivo images of large rats (up to 350 g), mice and human postmortem tissues were obtained to demonstrate coil functionality and to provide examples of potential applications at 21.1 T.

HIV-1 Capsid Function Is Regulated by Dynamics: Quantitative Atomic-Resolution Insights by Integrating Magic-Angle-Spinning NMR, QM/MM, and MD

HIV-1 CA capsid protein possesses intrinsic conformational flexibility, which is essential for its assembly into conical capsids and interactions with host factors. CA is dynamic in the assembled capsid, and residues in functionally important regions of the protein undergo motions spanning many decades of time scales. Chemical shift anisotropy (CSA) tensors, recorded in magic-angle-spinning NMR experiments, provide direct residue-specific probes of motions on nano- to microsecond time scales. We combined NMR, MD, and density-functional-theory calculations, to gain quantitative understanding of internal backbone dynamics in CA assemblies, and we found that the dynamically averaged 15N CSA tensors calculated by this joined protocol are in remarkable agreement with experiment. Thus, quantitative atomic-level understanding of the relationships between CSA tensors, local backbone structure, and motions in CA assemblies is achieved, demonstrating the power of integrating NMR experimental data and theory for characterizing atomic-resolution dynamics in biological systems.

A strategy for acquisition and analysis of complex natural abundance 33S solid-state NMR spectra of a disordered tetrathio transition-metal anion

A strategy, involving (i) sensitivity enhancement for the central transition (CT) by population transfer (PT) employing WURST inversion pulses to the satellite transitions (STs) in natural abundance (33)S MAS NMR for two different MAS frequencies (nu(r)=5.0 and 10.0kHz) at 14.1T and (ii) a (33)S static QCPMG experiment at 19.6T, has allowed acquisition and analysis of very complex solid-state (33)S CT NMR spectra for the disordered tetrathioperrhenate anion ReS(4)(-) in [(C(2)H(5))(4)N][ReS(4)]. This strategy of different NMR experiments combined with spectral analysis/simulations has allowed determination of precise values for two sets of quadrupole coupling parameters (C(Q) and eta(Q)) assigned to the two different S sites for the four sulfur atoms in the ReS(4)(-) anion in the ratio S1:S2=1:3. These sets of C(Q), eta(Q) values for the S1 and S2 site are quite similar and the magnitudes of the quadrupole coupling constants (C(Q)=2.2-2.5MHz) are a factor of about three larger than observed for other tetrathiometalates A(2)MS(4) (A=NH(4), Cs, Rb and M=W, Mo). In addition, the spectral analysis also leads to a determination of the chemical shift anisotropy (CSA) parameters (delta(sigma) and eta(sigma)) for the S1 and S2 site, however, with much lower precisions (about 20% error margins) compared to those for C(Q), eta(Q), because the magnitudes of the two CSAs (i.e., delta(sigma)=60-90ppm) are about a factor of six smaller than observed for the other tetrathiometalates mentioned above. This large difference in the magnitudes of the anisotropic parameters C(Q) and delta(sigma) for the ReS(4)(-) anion, compared to those for the WS(4)(2-) and MoS(4)(2-) anions determined previously under identical experimental conditions, accounts for the increased complexity of the PT-enhanced (33)S MAS spectra observed for the ReS(4)(-) anion in this study. This difference in C(Q) also contributes significantly to the intensity distortions observed in the outer wings of the CTs when employing PT from the STs under conditions of slow-speed MAS.

14N Polarization Inversion Spin Exchange at Magic Angle (PISEMA)

Polarization Inversion Spin Exchange at Magic Angle (PISEMA) is a powerful experiment for determining peptide orientation in uniformly aligned samples such as planar membranes. In this paper, we present (14)N-PISEMA experiment which correlates (14)N quadrupolar coupling and (14)N-(1)H dipolar coupling. (14)N-PISEMA enables the use of (14)N quadrupolar coupling tensor as an ultra sensitive probe for peptide orientation and can be carried out without the need of isotope enrichment. The experiment is based on selective spin-exchange between a proton and a single-quantum transition of (14)N spins. The spin-exchange dynamics is described and the experiment is demonstrated with a natural abundant N-acetyl valine crystal sample.

Hydrophobic Mismatch Drives the Interaction of E5 with the Transmembrane Segment of PDGF Receptor

The oncogenic E5 protein from bovine papillomavirus is a short (44 amino acids long) integral membrane protein that forms homodimers. It activates platelet-derived growth factor receptor (PDGFR) β in a ligand-independent manner by transmembrane helix-helix interactions. The nature of this recognition event remains elusive, as numerous mutations are tolerated in the E5 transmembrane segment, with the exception of one hydrogen-bonding residue. Here, we examined the conformation, stability, and alignment of the E5 protein in fluid lipid membranes of substantially varying bilayer thickness, in both the absence and presence of the PDGFR transmembrane segment. Quantitative synchrotron radiation circular dichroism analysis revealed a very long transmembrane helix for E5 of ∼26 amino acids. Oriented circular dichroism and solid-state 15N-NMR showed that the alignment and stability of this unusually long segment depend critically on the membrane thickness. When reconstituted alone in exceptionally thick DNPC lipid bilayers, the E5 helix was found to be inserted almost upright. In moderately thick bilayers (DErPC and DEiPC), it started to tilt and became slightly deformed, and finally it became aggregated in conventional DOPC, POPC, and DMPC membranes due to hydrophobic mismatch. On the other hand, when E5 was co-reconstituted with the transmembrane segment of PDGFR, it was able to tolerate even the most pronounced mismatch and was stabilized by binding to the receptor, which has the same hydrophobic length. As E5 is known to activate PDGFR within the thin membranes of the Golgi compartment, we suggest that the intrinsic hydrophobic mismatch of these two interaction partners drives them together. They seem to recognize each other by forming a closely packed bundle of mutually aligned transmembrane helices, which is further stabilized by a specific pair of hydrogen-bonding residues.

17O MAS NMR Correlation Spectroscopy at High Magnetic Fields

The structure of two protected amino acids, FMOC-l-leucine and FMOC-l-valine, and a dipeptide, N-acetyl-l-valyl-l-leucine (N-Ac-VL), were studied via one- and two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy. Utilizing 17O magic-angle spinning (MAS) NMR at multiple magnetic fields (17.6-35.2 T/750-1500 MHz for 1H) the 17O quadrupolar and chemical shift parameters were determined for the two oxygen sites of each FMOC-protected amino acids and the three distinct oxygen environments of the dipeptide. The one- and two-dimensional, 17O, 15N-17O, 13C-17O, and 1H-17O double-resonance correlation experiments performed on the uniformly 13C,15N and 70% 17O-labeled dipeptide prove the attainability of 17O as a probe for structure studies of biological systems. 15N-17O and 13C-17O distances were measured via one-dimensional REAPDOR and ZF-TEDOR experimental buildup curves and determined to be within 15% of previously reported distances, thus demonstrating the use of 17O NMR to quantitate interatomic distances in a fully labeled dipeptide. Through-space hydrogen bonding of N-Ac-VL was investigated by a two-dimensional 1H-detected 17O R3-R-INEPT experiment, furthering the importance of 17O for studies of structure in biomolecular solids.