Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins

Kay, Lewis E.; Ikura, Mitsuhiko; Tschudin, Rolf; Bax, Ad

doi:10.1016/j.jmr.2011.09.004

Cited by 277 publications

(224 citation statements)

References 36 publications

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“…PLN is a 52-residue transmembrane (TM) protein highly conserved across mammals (2). Its helix-loop-helix secondary structure is further subdivided into four dynamic domains: domain Ia (1-16), loop (17)(18)(19)(20)(21)(22), domain Ib (23)(24)(25)(26)(27)(28)(29)(30), and domain II (3,4). The hydrophobic TM domain II is the most conserved and responsible for SERCA inhibition, whereas the cytoplasmic domain harbors two phosphorylation sites that reverse PLN inhibitory function (2).…”

mentioning

confidence: 99%

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Verardi

Shi

Traaseth

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

155

210

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Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca 2þ -ATPase (SERCA), thereby regulating calcium homeostasis in cardiac muscle. In membranes, PLN forms pentamers that have been proposed to function either as a storage for active monomers or as ion channels. Here, we report the T-state structure of pentameric PLN solved by a hybrid solution and solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11°with respect to the membrane normal. The tilt angle difference between the monomer and pentamer is approximately 13°, showing that intramembrane helix-helix association forces dominate over the hydrophobic mismatch, driving the overall topology of the transmembrane assembly. Our data reveal that both topology and function of PLN are shaped by the interactions with lipids, which fine-tune the regulation of SERCA.hybrid NMR method | PISEMA | calcium regulation | oligomeric protein | dipolar assisted rotational resonance recoupling T he membrane protein complex formed by Ca 2þ -ATPase (SERCA) and phospholamban (PLN) regulates Ca 2þ concentration within the sarcoplasmic reticulum (SR), thereby controlling muscle excitation-contraction coupling (1, 2). PLN is a 52-residue transmembrane (TM) protein highly conserved across mammals (2). Its helix-loop-helix secondary structure is further subdivided into four dynamic domains: domain Ia (1-16), loop (17)(18)(19)(20)(21)(22), and domain II (31-52) (3, 4). The hydrophobic TM domain II is the most conserved and responsible for SERCA inhibition, whereas the cytoplasmic domain harbors two phosphorylation sites that reverse PLN inhibitory function (2). PLN has a direct role in the pathophysiology of the heart muscle, with three lethal mutations linked to dilated cardiomyopathy in humans (R9C-PLN, R14del, and L39-truncated-PLN) (5). In both synthetic and cell membranes, PLN forms pentamers that dissociate into monomers upon interacting with SERCA (1, 6). Although the stoichiometry of the SERCA/PLN complex has been assessed (1, 6), both the role and the structure of the PLN pentamer remain a matter of active debate. Because PLN expression in both atria and ventricles is higher than SERCA, it is likely that oligomerization may participate in SERCA regulation (7). Insights into PLN organization in the membrane have come from biochemical and biophysical data (2,6,8). Initial electrophysiological measurements indicated that PLN formed Ca 2þ channels (9). However, more recent electrochemical studies concluded that PLN does not conduct Cl − or Ca 2þ ions (10).Divergent structural models for the PLN pentamer have been proposed in the literature (8). Although very similar in the secondary structure content, these models differ in t...

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mentioning

confidence: 99%

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Verardi

Shi

Traaseth

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

155

210

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“…In cases where significant degeneracy is present in the 2D HSQC, threedimensional spectrum (such as HNCO or HNCA) may prove useful in resolving spin systems which overlap (Markley et al, 2003). The HNCO experiment can be used to predict secondary structure, and does so by correlating the amide 1 H and 15 N chemical shifts of one residue with the 13 CO chemical shift of the preceding residue (Grzesiek & Bax, 1993;Kay et al, 1990;Muhandiram & Kay, 1994). Here, magnetization is passed from 1 H to 15 N and to the 13 C by way of the 15 N _13 CO J coupling and then passed back via 15 N to 1 H for detection (refer to Fig.…”

Section: A Brief Introduction To Nmr Spectroscopymentioning

confidence: 99%

“…Here, magnetization is passed from 1 H to 15 N and to the 13 C by way of the 15 N _13 CO J coupling and then passed back via 15 N to 1 H for detection (refer to Fig. 4a); the chemical shift is evolved on all 3 nuclei which results in a threedimensional spectrum (Grzesiek & Bax, 1993;Kay et al, 1990;Muhandiram & Kay, 1994). HNCA, on the other hand, correlates the intraresidue 13 C chemical shift with the amide 1 H and 15 N shifts (Farmer et al, 1992;Grzesiek & Bax, 1993;Kay et al, 1990).…”

Section: A Brief Introduction To Nmr Spectroscopymentioning

confidence: 99%

Structure and Dynamics of Proteins from Nuclear Magnetic Resonance Spectroscopy

Valafar¹,

Irausquin²

2012

Protein Structure

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“…Development of 15 N-labeling methodologies during the late 1980s improved peak dispersion, enabling the assignment of larger protein molecules using the sequential assignment approach [4], [5]. [6], [7]. This triple-resonance strategy relies on set of NMR experiments that utilize through-bond (spin-spin) couplings to identify spin systems that belong to either single amino acid or dipeptide.…”

Section: Solution-state Nmr Sequential Protein Resonance Manual Assigmentioning

confidence: 99%

“…As a result, uncompressed JSONized NMR-STAR files occupy more disk space (Table 6). However, the nmrstarlib library offers the ability to read NMR-STAR files in both uncompressed (directory of files) and > # print saveframe names > names(starfile) [1] "data" "save_entry_information" [3] "save_entry_citation" "save_assembly" [5] "save_EVH1" "save_natural_source" [7] "save_experimental_source" "save_sample_1" [9] "save_sample_2" "save_sample_3" [11] "save_sample_4" "save_sample_conditions_1" [13] "save_sample_conditions_2" "save_sample_conditions_3" [15] "save_sample_conditions_4" "save_AZARA" [17] "save_xwinnmr" "save_ANSIG" [19] "save_CNS" "save_spectrometer_1" [21] "save_spectrometer_2" "save_NMR_spectrometer_list" [23] "save_experiment_list" "save_chemical_shift_reference_1" [25] "save_assigned_chem_shift_list_1" "save_combined_NOESY_peak_list" > # access saveframe key-value data > starfile$data [1] Figure 27. Code example showing how to access data from JSONized NMR-STAR files using C++ programming language.…”

Section: Advantages Of Using Nmrstarlib and Jsonized Nmr-star Versionmentioning

confidence: 99%

Algorithms for automated assignment of solution-state and solid-state protein NMR spectra.

Smelter¹

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Protein nuclear magnetic resonance spectroscopy (Protein NMR) is an invaluable analytical technique for studying protein structure, function, and dynamics. There are two major types of NMR spectroscopy that are used for investigation of protein structuresolution-state and solid-state NMR. Solution-based NMR spectroscopy is typically applied to proteins of small and medium size that are soluble in water. Solid-state NMR spectroscopy is amenable for proteins that are insoluble in water.In the vast majority NMR-based protein studies, the first step after experiment optimization is the assignment of protein resonances via the association of chemical shift values to specific atoms in a protein macromolecule. Depending on the quality of the spectra, a manual protein resonance assignment process often requires a considerable amount of time, from weeks to months-worth of effort even, by an experienced NMR spectroscopist.vii The resonance assignment processes for solution-state and solid-state protein NMR studies are conceptually similar, but have distinct differences due to the utilization of different NMR experiments and to the use of different resonances for grouping peaks into spin systems.Currently, there is a shortage of robust, effective software tools that can perform solid-state protein resonance assignment and there is no general software that can perform both solution-state and solid-state protein resonance assignment in a reliable, automated fashion. Hence, the motivation of this research is to design and implement algorithms and software tools that will automate the resonance assignment problem.As a result of this research, several algorithms and software packages that aid several important steps in the protein resonance assignment process were developed. For example, the nmrstarlib software package can access and utilize data deposited in the NMR-STAR format; the core of this library is the lexical analyzer for NMR-STAR syntax that acts as a generator-based state-machine for token processing. The jpredapi software package provides an easy-to-use API to submit and retrieve results from secondary structure prediction server. The single peak list and pairwise peak list registration algorithms address the problem of multiple sources of variance within single peak list and between different peak lists and is capable of calculating the match tolerance values necessary for spin system grouping. The single peak list and pairwise peak list grouping algorithms are based on the well-known DBSCAN clustering algorithm and are designed to group peaks into spin systems within single peak list as well as between different peak lists.viii

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Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins

Cited by 277 publications

References 36 publications

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Structure and Dynamics of Proteins from Nuclear Magnetic Resonance Spectroscopy

Algorithms for automated assignment of solution-state and solid-state protein NMR spectra.

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