Papers by Peter L Gor'kov
Journal of Biomolecular NMR, Oct 7, 2011
Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane pro... more 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 (1)H-(15)N dipolar couplings (DC) and (15)N 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 ([(1)H,(15)N]-SE-PISEMA-PDSD). The incorporation of 2D (15)N/(15)N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the (15)N 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.
Journal of the American Chemical Society, Oct 18, 2016
HIV-1 CA capsid protein possesses intrinsic conformational flexibility, which is essential for it... more 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 timescales. Chemical shift anisotropy (CSA) tensors, recorded in magic-anglespinning NMR experiments, provide direct residue-specific probes of motions on nano-to
Journal of Physical Chemistry C, Jul 7, 2022
Journal of the American Chemical Society, Oct 10, 2022
Solid State Nuclear Magnetic Resonance, Feb 1, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Journal of Chemical Physics, Oct 21, 2019
Indirect detection via sensitive spin-1/2 nuclei like protons under magic-angle spinning (MAS) ha... more Indirect detection via sensitive spin-1/2 nuclei like protons under magic-angle spinning (MAS) has been developed to overcome the low spectral sensitivity and resolution of 14 N NMR. The 14 N quadrupolar couplings cause inefficient encoding of the 14 N frequency due to large frequency offsets and make the rotor-synchronization of the evolution time necessary. It is shown that 14 N rf pulses longer than the rotor period can efficiently encode 14 N frequencies and generate spinning sideband free spectra along the indirect dimension. Average Hamiltonian and Floquet theories in the quadrupolar jolting frame (QJF) are used to treat the spin dynamics of the spin-1 quadrupolar nucleus under long 14 N rf pulses and MAS. The results show that the rf action can be described by a scaled and phase-shifted effective rf field. The large quadrupolar frequency offset becomes absent in the QJF, therefore leads to sideband-free spectra along the indirect dimension. More importantly when a pair of long 14 N rf pulses are used, the distribution of the phase shift of the effective rf field does not affect the 14 N encoding for powder samples, thus high efficiencies can be obtained. The efficient and sideband-free features are demonstrated for three 1 H/ 14 N indirectly-detected experiments using long 14 N pulses under fast MAS.
Journal of the American Chemical Society, Nov 13, 2013
A key stage in HIV-1 maturation towards an infectious virion requires sequential proteolytic clea... more A key stage in HIV-1 maturation towards 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 intermediates using Magic Angle Spinning NMR spectroscopy. At magnetic fields of 19.9 T and above, outstandingquality 2D and 3D MAS NMR spectra were obtained for tubular CA and CA-SP1 assemblies yield, permitting resonance assignments for subsequent detailed structural characterization.
Chemistry: A European Journal, May 27, 2020
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Proceedings of the National Academy of Sciences of the United States of America, Nov 9, 2015
The structure of two protected amino acids, FMOC-l-leucine and FMOC-l-valine, and a dipeptide, N-... more 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.National Institutes of Health (U.S.) (Grant EB-001960)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-002026
Journal of Magnetic Resonance, 2009
Polarization Inversion Spin Exchange at Magic Angle (PISEMA) is a powerful experiment for determi... more 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 spinexchange dynamics is described and the experiment is demonstrated with a natural abundant N-acetyl valine crystal sample.
Journal of Magnetic Resonance, Jul 1, 2006
15 N detection of mechanically aligned membrane proteins benefits from large sample volumes that ... more 15 N detection of mechanically aligned membrane proteins benefits from large sample volumes that compensate for the low sensitivity of the observe nuclei, dilute sample preparation, and for the poor filling factor arising from the presence of alignment plates. Use of larger multi-tuned solenoids, however, is limited by wavelength effects that lead to inhomogeneous RF fields across the sample, complicating cross-polarization experiments. We describe a 600 MHz 15 N-1 H solid-state NMR probe with large (580 mm 3) RF solenoid for high-power, multi-pulse sequence experiments, such as polarization inversion spin exchange at the magic angle (PISEMA). In order to provide efficient detection for 15 N, a 4-turn solenoidal sample coil is used that exceeds 0.27k at the 600 MHz 1 H resonance. A balanced tuning-matching circuit is employed to preserve RF homogeneity across the sample for adequate magnetization transfer from 1 H to 15 N. We describe a procedure for optimization of the shorted 1/4k coaxial trap that allows for the sufficiently strong RF fields in both 1 H and 15 N channels to be achieved within the power limits of 300 W 1 H and 1 kW 15 N amplifiers. The 8 • 6 • 12 mm solenoid sustains simultaneous B 1 irradiation of 100 kHz at 1 H frequency and 51 kHz at 15 N frequency for at least 5 ms with 265 and 700 W of input power in the respective channels. The probe functionality is demonstrated by 2D 15 N-1 H PISEMA spectroscopy for two applications at 600 MHz.
Biochemistry, Aug 18, 2006
Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two i... more Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca 2+ affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares ∼30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively 15 N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D 15 N PISEMA experiments. The PISEMA spectra obtained on uniformly 15 N-labeled protein as well as 15 N-Leu, 15 N-Ile and 15 N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by ∼23°with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by ∼5°. The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.
Journal of Magnetic Resonance, Mar 1, 2007
RF heating of solid-state biological samples is known to be a destabilizing factor in high-field ... more RF heating of solid-state biological samples is known to be a destabilizing factor in high-field NMR experiments that shortens the sample lifetime by continuous dehydration during the high-power cross-polarization and decoupling pulses. In this work, we describe specially designed, large volume, low-E 15 N-1 H solid-state NMR probes developed for 600 and 900 MHz PISEMA studies of dilute membrane proteins oriented in hydrated and dielectrically lossy lipid bilayers. The probes use an orthogonal coil design in which separate resonators pursue their own aims at the respective frequencies, resulting in a simplified and more efficient matching network. Sample heating at the 1 H frequency is minimized by a loop-gap resonator which produces a homogeneous magnetic field B 1 with low electric field E. Within the loop-gap resonator, a multi-turn solenoid closely matching the shape of the sample serves as an efficient observe coil. We compare power dissipation in a typical lossy bilayer sample in the new low-E probe and in a previously reported 15 N-1 H probe which uses a double-tuned 4-turn solenoid. RF loss in the sample is measured in each probe by observing changes in the 1 H 360°pulse lengths. For the same values of 1 H B 1 field, sample heating in the new probe was found to be smaller by an order of magnitude. Applications of the low-E design to the PISEMA study of membrane proteins in their native hydrated bilayer environment are demonstrated at 600 and 900 MHz.
Journal of Magnetic Resonance, Sep 1, 2009
A balanced probe circuit is used to generate high B 1 magnetic field for sensitivity enhancement ... more 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-c quadrupolar nuclei. Electrical balancing of the sample coil can cut the peak voltage by a half, therefore improving the power handling when generating a twofold 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-c quadrupolar nuclei such as 25 Mg at natural abundance.
Journal of the American Chemical Society, Apr 4, 2007
High-resolution solid-state NMR spectra of three full-length membrane proteins uniformly aligned ... more High-resolution solid-state NMR spectra of three full-length membrane proteins uniformly aligned in lipid bilayers between glass slides are observed at high magnetic field. The resolution of the specific amino acid labeled samples shows promise for large membrane protein structure determination utilizing aligned samples and shows resonance patterns known as PISA wheels. The tilt angles of the transmembrane helices are extracted from the resonance patterns in PISEMA spectra. Progress has been made in membrane protein structure determination in lipid bilayer environments 1. From the complete structure of gramicidin in 1993 1g to the structure of the M2 transmembrane domain with 2 and without 1e the antiviral drug amantadine, the structure of MerFt 1a , etc., there are now ten structures in the Protein Data Bank characterized by aligned sample solid state NMR. Recent improvements in RF probe technology 3 and in sample preparation have make it possible here to obtain spectra of uniformly aligned full-length membrane proteins displaying characteristic resonance patterns for their transmembrane (TM) α-helices 4. Sample preparation is the key to macromolecular structural characterization, whether it is crystallization for x-ray diffraction or cryo-EM, or homogeneous isotropic samples for solution NMR, or a uniformly aligned sample for solid state NMR. Here, from monomeric to octameric, from 3.5 to 82 kDa and from one to three TM helices per monomer we demonstrate uniform protein alignment in hydrated phospholipid bilayers on glass slides. The helical structure prediction for three proteins is shown in Fig. 1. KdpF is a 30 residue protein (33 residues as expressed in E. coli) from the Mycobacterium tuberculosis genome. This protein appears to be a component of the Kdp K + transporting complex 8. It has a single putative TM helix and no predicted 2° structure for the terminal segments. Rv1861 is another
Journal of the American Chemical Society, Apr 30, 2002
Protein Science, Nov 1, 2007
For the first time, 15 N solid-state NMR experiments were conducted on wild-type phospholamban (W... more For the first time, 15 N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15 N solid-state NMR spectra of site-specific 15 N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13°6 6°with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from sitespecific 15 N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/ 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.
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Papers by Peter L Gor'kov