Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions

Piotto, Martial; Saudek, Vladimı́r; Sklenář, Vladimı́r

doi:10.1007/bf02192855

Cited by 3,635 publications

(3,230 citation statements)

References 19 publications

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“…The 'H a pulse in the second INEPT step was replaced by a WATER-GATE 3-9-19 pulse train (xl8.65, A, x/2.88, A, x/1.37, A, x/1.37, A, al2.88, A, a/8.65;, A = 160 ps) flanked by two gradients (G3, I ms, 17.5 gausslcm) (Piotto et al, 1992). CARP decoupling was applied on the "N channel during acquisition (Shaka et al, 1985).…”

Section: Identification Of the Imidazole 'H And "N Resonances Of His 49mentioning

confidence: 99%

“…Water suppression was achieved using the WATER-GATE sequence, in which the final nonselective 180" ' H pulse in the standard 'H-"N HSQC sequence was also replaced with a composite (3-9-19) 180" pulse that is antiselective for the water resonance and is sandwiched between two gradient pulses, the purpose of which is to defocus the residual water magnetization (Piotto et al, 1992) (Fig. 5 ) .…”

Section: Measurement Of Apparent Amide Proton T Relaxation Ratesmentioning

confidence: 99%

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Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

Lyons¹,

Ratnaswamy²,

Pochapsky³

1996

Protein Science

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Multidimensional NMR methods were used to obtain 'H-I'N correlations and "N resonance assignments for amide and side-chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P-450,,, (CYPlOl). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 33:6424-6432) and redox-dependent ' H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry33:6433-6441). "N assignments were made with NOESY-('H/''N) HMQC and TOCSY-('H/''N) HSQC spectra obtained using samples of Pdx uniformly labeled with "N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue-specific amide proton exchange rates, which were measured by a combination of saturation transfer and H 2 0 / D 2 0 exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation-state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for residues near the metal center and in a region of compact structure that includes the loop-turn Val 74-Ser 82 and the C-terminal residues (Pro 102-Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction between Pdx and CYPlOl upon the oxidation state of Pdx.

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Section: Identification Of the Imidazole 'H And "N Resonances Of His 49mentioning

confidence: 99%

Section: Measurement Of Apparent Amide Proton T Relaxation Ratesmentioning

confidence: 99%

Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

Lyons¹,

Ratnaswamy²,

Pochapsky³

1996

Protein Science

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“…A reference 1D proton spectrum was taken in H,O buffer at 20.0 "C. The water resonance was suppressed by the non-saturative WATERGATE technique (Piotto et al, 1992). Within 25 min of the resuspension, data collection had begun at 20.0 "C. Forty-eight individual 1D proton spectra were collected in a continuous fashion over a total time of 16 h; each spectrum took 20 min to collect.…”

Section: Amide Exchangementioning

confidence: 99%

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Boice¹,

Fairman²

1996

Protein Science

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The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein-protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin-like repeats, motifs implicated in protein-protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self-associates to form dimers with a Kd = 270 pM. The CD spectrum indicates that the protein is composed of 33% a-helix, 22% P-sheet, 19% p-turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a AG of -2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogendeuterium exchange, and fluorescence. It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.Keywords: analytical ultracentrifugation; ankyrin-like repeat; cyclin; circular dichroism; cyclin dependent kinase inhibitor Control of the eukaryotic cell cycle is a highly regulated process that involves the interaction of a large number of macromolecules. Critical checkpoints in this process are found during the entry of cells into both the GUS and G2/M phases of the cell cycle (for reviews, see Draetta, 1994;Sherr, 1995). The cyclin-dependent kinases (CDKs) are enzymes whose activities govern these checkpoints. CDK holoenzymes, which consist of a CDK catalytic subunit and a cyclin regulatory subunit, are regulated, in part, by their temporal expression, degradation, phosphorylation, and dephosphorylation. This regulation throughout the cell cycle serves to drive the cell through the successive phases.Recent studies have identified CDK inhibitors, a new class of proteins, adding yet another level of control to CDK regulation (for review, see Elledge & Harper, 1994). These CDK inhibitors are thought to function by blocking and/or disrupting CDK-cyclin association. p16, one of the first of these inhibitors to be discovered, was originally cloned from a yeast-two hybrid interaction screen using CDK4 as the bait protein (Serrano et al., 1993).

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“…The remainder of the purification of 15 N myristoylated NCS-1 was the same as described above. Two-dimensional 1 H-15 N heteronuclear single quantum correlation (HSQC) experiments were collected at either 800 MHz on a Varian INOVA spectrometer or at 600 MHz on a Bruker Avance II spectrometer outfitted with a coldprobe at 298 K using WATERGATE for solvent suppression [23]. A uniformly 15 N -labeled myristoylated NCS-1 sample was prepared by extensive buffer exchange into 50 mM Tris, 4mM CaCl 2 , 100 mM KCl, 5 mM DTT with a pH of 7.2 with no correction following the addition of 10% D 2 O.…”

Section: Expression Of 15 N Myristoylated Ncs-1 and Nmr Spectroscopymentioning

confidence: 99%

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

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We have developed a protocol to produce large quantities of high purity myristoylated and nonmyristoylated Neuronal Calcium Sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6X His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100 -200 µM). With optimized growth conditions and a high capacity metal affinity purification scheme, > 50 mg of homogenous myristoylated NCS-1 is obtained from 1 L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 EF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.

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Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions

Cited by 3,635 publications

References 19 publications

Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

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