Abstract:
:The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.
journal_name
J Biomol NMRjournal_title
Journal of biomolecular NMRauthors
Antanasijevic A,Kingsley C,Basu A,Bowlin TL,Rong L,Caffrey Mdoi
10.1007/s10858-016-0025-1subject
Has Abstractpub_date
2016-03-01 00:00:00pages
255-65issue
3eissn
0925-2738issn
1573-5001pii
10.1007/s10858-016-0025-1journal_volume
64pub_type
杂志文章abstract::One of the major bottlenecks in the determination of proteinstructures by NMR is in the evaluation of the data produced by theexperiments. An important step in this process is assignment, where thepeaks in the spectra are assigned to specific spins within specificresidues. In this paper, we discuss a spin system assig...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1023/A:1018329420659
更新日期:1997-10-01 00:00:00
abstract::Sparse sampling in biomolecular multidimensional NMR offers increased acquisition speed and resolution and, if appropriate conditions are met, an increase in sensitivity. Sparse sampling of indirectly detected time domains combined with the direct truly multidimensional Fourier transform has elicited particular attent...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-011-9584-3
更新日期:2012-01-01 00:00:00
abstract::The cryogenic temperatures at which dynamic nuclear polarization (DNP) solid-state NMR experiments need to be carried out cause line-broadening, an effect that is especially detrimental for crowded protein spectra. By increasing the magnetic field strength from 600 to 800 MHz, the resolution of DNP spectra of type III...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-016-0044-y
更新日期:2016-08-01 00:00:00
abstract::A new homonuclear Hartmann-Hahn-type mixing scheme is introduced that effects coherence transfer between resonances in two separated frequency bands. The mixing scheme relies on the irradiation of two-band selective shaped pulses that are expanded in an MLEV-16 supercycle. Similar to heteronuclear Hartmann-Hahn experi...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/BF00211162
更新日期:1996-09-01 00:00:00
abstract::Temperature coefficients have been measured for backbone amide (1)H and (15)N nuclei in the B1 domain of protein G (GB1), using temperatures in the range 283-313 K, and pH values from 2.0 to 9.0. Many nuclei display pH-dependent coefficients, which were fitted to one or two pK(a) values. (1)H coefficients showed the e...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-011-9583-4
更新日期:2012-01-01 00:00:00
abstract::Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR s...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-006-9070-5
更新日期:2006-09-01 00:00:00
abstract::Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-019-00235-7
更新日期:2019-09-01 00:00:00
abstract::Two methods for the measurement of homonuclear 3J(HNH)alpha coupling constants are described. Both HSQC- and HMQC-type experiments employ 'quantitative J-correlation', in which the coupling constant of interest is obtained from the intensity ratio of cross peaks of two spectra. The first spectrum is acquired with 3J(H...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1023/a:1008343926502
更新日期:2000-01-01 00:00:00
abstract::A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue 'i' with that of residues 'i-1' and 'i+1' in ((13)C, (15)N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploi...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-015-9976-x
更新日期:2015-10-01 00:00:00
abstract::1H(i)-15 N(i)-13C'(i) dipole-chemical shift anisotropy (CSA) relaxation interference was quantified for the 13C,15N labeled zinc-finger protein qCRP2(LIM2). The cross-correlation rates obtained for residues located in the metal coordination sites of qCRP2(LIM2) show a high degree of correlation with the peptide plane ...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1023/a:1011248220839
更新日期:2001-04-01 00:00:00
abstract::We present and test two methods to use quantum chemical calculations to improve standard protein structure refinement by molecular dynamics simulations restrained to experimental NMR data. In the first, we replace the molecular mechanics force field (employed in standard refinement to supplement experimental data) for...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-004-6729-7
更新日期:2005-02-01 00:00:00
abstract::The human alpha 3-chain type VI collagen C-terminal Kunitz domain fragment (alpha 3(VI)) has been studied by two dimensional 1H-1H and 1H-13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and a number of unusual H alpha chemical shifts, which ...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/BF00228142
更新日期:1996-12-01 00:00:00
abstract::Extensive resonance overlap exacerbates assignment of intrinsically disordered proteins (IDPs). This issue can be circumvented by utilizing (15)N, (13)C' and (1)H(N) spins, where the chemical shift dispersion is mainly dictated by the characteristics of consecutive amino acid residues. Especially (15)N and (13)C' spin...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-011-9470-z
更新日期:2011-02-01 00:00:00
abstract::A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gain...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-007-9149-7
更新日期:2007-05-01 00:00:00
abstract::Great theoretical and methodological advances are pushing the limits of resolution and sensitivity in solid state NMR (SSNMR). However, sample preparation remains a critical issue for the success of an experiment. The factors affecting spectral quality in SSNMR samples are discussed, examining cases encountered in the...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-013-9776-0
更新日期:2013-10-01 00:00:00
abstract::Three experiments are introduced to determine a complete set of coupling constants in RNA oligomers. In the HCCH-E.COSY experiment, the vicinal proton-proton coupling constants can be measured with high accuracy. In the P-FIDS-CT-HSQC experiment, vicinal proton-phosphorus and carbon-phosphorus couplings are measured t...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/BF00404274
更新日期:1994-09-01 00:00:00
abstract::A substantial time savings in the collection of multidimensional NMR data can be achieved by coupling the evolution of nuclei in the indirect dimensions. In order to save time, the sampling of the indirect dimensions is inherently incomplete. Therefore, many algorithms and samplings schemes have been developed aimed a...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-009-9309-z
更新日期:2009-05-01 00:00:00
abstract::Dynamics of large-amplitude conformational motions in proteins are complex and less understood, although these processes are intimately associated with structure, folding, stability, and function of proteins. Here, we use a large set of spectra obtained by cross-relaxation suppressed exchange NMR spectroscopy (EXSY) t...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-007-9186-2
更新日期:2007-11-01 00:00:00
abstract::The discovery of the TROSY effect (Pervushin et al. in Proc Natl Acad Sci USA 94:12366-12371, 1997) for reducing transverse relaxation and line sharpening through selecting pathways in which dipole-dipole and CSA Hamiltonians partially cancel each other had a tremendous impact on solution NMR studies of macromolecules...
journal_title:Journal of biomolecular NMR
pub_type: 信件
doi:10.1007/s10858-016-0075-4
更新日期:2016-12-01 00:00:00
abstract::Complex mixtures are at the heart of biology, and biomacromolecules almost always exhibit their function in a mixture, e.g., the mode of action for a spider venom is typically dependent on a cocktail of compounds, not just the protein. Information about diseases is encoded in body fluids such as urine and plasma in th...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-013-9752-8
更新日期:2014-04-01 00:00:00
abstract::Senktide is a highly specific and potent analog of neurokinin B, the natural ligand of the tachykinin receptor NK-3. The membrane-bound conformation of senktide, interacting with negatively charged membrane vesicles composed of perdeuterated phosphatidylcholine and phosphatidylglycerol (70:30), has been investigated u...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/BF00176010
更新日期:1993-07-01 00:00:00
abstract::The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-020-00310-4
更新日期:2020-05-01 00:00:00
abstract::Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein-protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-018-0165-6
更新日期:2018-03-01 00:00:00
abstract::We present heteronuclear three-dimensional gradient-NMR techniques for the resonanceassignment of exchangeable (-OH and -NH) protons in uniformly 13C isotopically enrichedoligosaccharides and for the measurement of 1H-1H nuclear Overhauser enhancementsinvolving these protons. These techniques are derived from conventi...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1023/A:1018671517876
更新日期:1997-01-01 00:00:00
abstract::Nearly complete backbone 1H, 15N and 13C signal assignments are reported for beta-hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although 15N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experimen...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/BF00200435
更新日期:1996-06-01 00:00:00
abstract::Fast multidimensional NMR with a time resolution of a few seconds provides a new tool for high throughput screening and site-resolved real-time studies of kinetic molecular processes by NMR. Recently we have demonstrated the feasibility to record protein 1H-15N correlation spectra in a few seconds of acquisition time ...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-005-4425-x
更新日期:2005-12-01 00:00:00
abstract::For the understanding of cellular processes the molecular structure of biomolecules has to be accurately determined. Initial models can be significantly improved by structure refinement techniques. Here, we present the refinement methods and analysis techniques implemented in the GROMOS software for biomolecular simul...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-011-9534-0
更新日期:2011-11-01 00:00:00
abstract::Detection of (15)N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached (15)N nuclei (TROSY (15)NH) yields high quality s...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-015-9991-y
更新日期:2015-12-01 00:00:00
abstract::The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH ...
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-018-0197-y
更新日期:2018-06-01 00:00:00
abstract::We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T 2' times and a site-specific comparison of T 2' at 93 kHz versus 60 kHz MAS frequency....
journal_title:Journal of biomolecular NMR
pub_type: 杂志文章
doi:10.1007/s10858-015-9975-y
更新日期:2015-10-01 00:00:00