Minimizing Sensitivity Losses in Gradient-Selected 15N-1H HSQC Spectra of Proteins

Stonehouse, Jonathan; Shaw, Graeme L.; Keeler, James; Laue, Ernest D.

doi:10.1006/jmra.1994.1066

Cited by 121 publications

(87 citation statements)

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“…When possible, NMR data were acquired using sensitivity enhancement (Cavanagh & Rance, 1990;Kay et al, 1992) and pulsed-®eld gradients for coherence transfer pathway selection and artifact suppression (Kay, 1995;Keeler et al, 1994). Sensitivity was further improved by minimizing the degree of water saturation (Grzesiek & Bax, 1993;Kay et al, 1994;Li & Montelione, 1993;Stonehouse et al, 1994). Two-dimensional 1 H-15 N HSQC spectra (Bodenhausen & Ruben, 1979;Zhang et al, 1994) (Figure 1) were acquired with 128 complex points in t 1 , 1024 complex points in t 2 and 16 transients.…”

Section: Nmr Data Acquisitionmentioning

confidence: 99%

Equilibrium folding intermediates of a greek key β-barrel protein

Bagby

Inouye

et al. 1998

Journal of Molecular Biology

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Protein S is a calcium-binding protein comprising two Greek key b-barrel domains. We have used NMR and optical spectroscopies to show that, in the absence of calcium, the N-terminal domain of protein S forms two equilibrium folding intermediates that are in slow exchange. The intermediates arise from differential calcium-dependent folding of subdomains which are not contiguous along the polypeptide chain. The structures of these intermediates are incompatible with several previously proposed folding mechanisms for Greek key b-barrel domains. We propose a different mechanism that involves multiple nucleation sites for folding and sequential acquisition of native long-range interactions.

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Section: Nmr Data Acquisitionmentioning

confidence: 99%

Equilibrium folding intermediates of a greek key β-barrel protein

Bagby

Inouye

et al. 1998

Journal of Molecular Biology

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“…Finally, the resonances merge with the water peak in the fast exchange limit. The effect of slow or intermediate exchange with water on the intensity of HSQC peaks can be minimized by using experiments which do, not perturb the water resonance (Grzesiek & Bax, 1993;Stonehouse et al, 1994;Zhang et al, 1994) even at pH values slightly above neutral. The arginine side-chain guanidinium resonances were examined as a function of pH to probe the extent to which the different guanidinium side chains are protected from exchange by hydrogen bonding or burial in the PLCC SH2 domain-pY1021 complex.…”

Section: Amide Exchange With Solventmentioning

confidence: 99%

pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pK_a values

Singer

Forman‐Kay

1997

Protein Science

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Electrostatic interactions in a complex of the phospholipase C-y, C-terminal SH2 domain with a high-affinity binding phosphopeptide representing the sequence around Tyr 1021 of the p platelet-derived growth factor receptor were studied by pK<, determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His PD4) and the phosphotyrosine (pTyrj phosphate group show pK', values significantly lower than average for these residue types in proteins. The reduced pK, of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pK, of His PD4 is also due to burial from solvent in a hydrogen-bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pK, values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C-terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain-phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein.Keywords: histidine; NMR; phospholipase C-y; phosphotyrosine; pK, determination; SH2 domain Electrostatic interactions in proteins are very important in catalysis, ligand recognition processes, and protein stability (Gilson, 1995). We are interested in the electrostatic interactions of Src homology 2 (SH2) domains with their phosphotyrosine-containing targets. SH2 domains are small ("100 amino acid) domains that play an important role in signal transduction by binding to specific sequences on growth factor receptors or other molecules containing phosphotyrosine (for a review, see Schaffhausen, 1995). Studies of the binding of SH2 domains to their target proteins have been facilitated by modeling of the interacting region of the target protein with small (-20 amino acids or less) phosphopeptides containing sequences around the site of Tyr phosphorylation. These peptides can significantly inhibit the biological interaction (Yonezawa et al., 1992;Larose et al., 1993;Nishimura et al., 1993). SH2 domain binding to phosphopeptides is characterized by a very rapid (diffusion-limited or faster) on-rate (Panayotou et al., 1993), most likely due to electrostatic attraction of the phosphotyrosine (pTyr) phosphate groups with the highly positively charged binding pockets observed in the structures of SH2 domains and SH2 domain-phosphopeptide complexes solved to date (Eck et al., 1993;Waksman et al., 1993;Gilmer

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“…2D NMR spectra were acquired between 290 and 308 K in phase sensitive mode (TPPI). Water suppression was achieved either with WATERGATE [12] or, for hetero-nuclear experiments, with the pulse-sequence suggested by Stonehouse et al [13]. 2D and 3D TOCSY spectra were acquired using the TOWNY composite pulse cycle [14].…”

Section: Nuclear Magnetic Resonancementioning

confidence: 99%

“…Since the solubility of the sample after lyophilization decrease drastically, the degree of exposure of the amides was estimated from solvent saturation experiments. Two HSQC were performed in the absence and in the presence of water presaturation using the same receiver gain (the water was suppressed anyway by gradients either according to [12] or [13]). Peaks were integrated by the UXNMR software and they were considered protected when the percentage ratio of the values obtained in the presence and in the absence of solvent saturation was higher then 50% normalized on the least affected peak.…”

Section: Nuclear Magnetic Resonancementioning

confidence: 99%

“…Since they are in unstructured loop regions, their amide protons are subjected to significant loss of sensitivity, which is especially high at neutral pH. This effect could be reduced but not cancelled using pulse field gradient techniques [12,13]. Ala-30, Ser-49 and Glu-50 could however be observed from their c~ protons in the TOCSY and tentatively assigned.…”

Section: Ma Castiglione Morelli Et Al/febs Letters 358mentioning

confidence: 99%

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The KH module has an αβ fold

Morelli¹,

Stier²,

Gibson³

et al. 1995

FEBS Letters

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The KH module has recently been identified in a number of RNA associated proteins including vigilin and FMR1, a protein implicated in the fragile X syndrome. In this work, NMR spectroscopy was used to determine the secondary structure in solution of a KH domain (repeat 5 from vigilin). Almost complete assignments were obtained for the ~H and ~SN resonances using uniform ~SN-labeling of the protein combined with homo-nuclear 2D ~HNMR and 3D lSN correlated ~H NMR. On the basis of NOE patterns, secondary chemical shifts and amide solvent exposure, the secondary structure consists of an antiparallel three stranded fl sheet connected by two helical regions. This domain may also be stabilized by an appended C-terminal helix which is common to many but not all members of the KH family.

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Minimizing Sensitivity Losses in Gradient-Selected 15N-1H HSQC Spectra of Proteins

Cited by 121 publications

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Equilibrium folding intermediates of a greek key β-barrel protein

Equilibrium folding intermediates of a greek key β-barrel protein

pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pK_a values

The KH module has an αβ fold

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Minimizing Sensitivity Losses in Gradient-Selected 15N-1H HSQC Spectra of Proteins

Cited by 121 publications

References 0 publications

Equilibrium folding intermediates of a greek key β-barrel protein

Equilibrium folding intermediates of a greek key β-barrel protein

pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pKa values

The KH module has an αβ fold

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pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pK_a values