Low-Conductivity Buffers for High-Sensitivity NMR Measurements

Kelly, Alexander E.; Ou, Horng D.; Withers, R.S.; Dötsch, Volker

doi:10.1021/ja026121b

Cited by 161 publications

(136 citation statements)

References 24 publications

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“…To address the reported discrepancy, we noticed that the buffer conditions used in the studies by Heuer and coworkers (37) contained 20 mM free inorganic phosphate. Given the moderate affinity of the ADAP SH3c-SKAP-55 peptide interaction when isolated in vitro, high conductivity buffers incorporating phosphate may well mask interactions that can be detected by NMR with greater sensitivity in low conductivity buffers (38). Phosphate in NMR buffer can interfere with SH2/ SH3 domain-ligand interactions.…”

Section: Discussionmentioning

confidence: 99%

Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein

Duke‐Cohan

Kang

Liu

et al. 2006

Journal of Biological Chemistry

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Modular domains in proteins such as protein-tyrosine kinases, phosphatases and adaptors play central roles in the generation of signals needed for mammalian cell function (1). Of these, Src homology domain 2 (SH2) 3 recognizes phosphotyrosine-based motifs, whereas Src homology domain 3 (SH3) domains recognize proline-based PXXP motifs (2). Since the description of Abelson SH3 domain recognition of the 3BP1 protein (3), numerous SH3 domain-mediated interactions have been documented. Examples include SH3-mediated interactions between the adaptor Grb-2 (growth factor receptor-bound protein-2) and Son-of-Sevenless, Src kinase SH3 domain binding to the p85 subunit of phosphatidylinositol 3-kinase, and Crk SH3 domain binding to Crk SH3 domain-binding guanine nucleotide-releasing factor, among others. SH3 domain binding is involved in subcellular localization, cytoskeletal organization, and signal transduction (4).Structurally, SH3 domains are comprised of two anti-parallel ␤ sheets packed at right angles to one other (2, 4, 5). The core øPXøP motif (where ø represents a hydrophobic residue) interacts with SH3 through two defined consensus sequences: Class I (R/KXXPXXP) and Class II (PXXPXR) (6). Domains can bind ligands in either an N-to C-terminal or C-to N-terminal orientation due to the pseudosymmetrical nature of the polyproline class II helix that is stabilized primarily by hydrophobic and additional electrostatic interactions. Directionality is conferred by the interaction of the arginine or lysine residues with the charged outer face on the SH3 domain, while the tandem prolines bind to two hydrophobic pockets. Binding depends on the two SH3 variable loops, the RT and n-Src loops, that flank a ligand-binding region.In addition to the binding to øPXøP-based motifs, an increasing number of studies have documented SH3 domain binding to non-canonical motifs (7). These include a PX(V/I)(D/N)RXXKP motif that is responsible for STAM2 SH3 domain binding to the deubiquitination enzyme ubiquitin isopeptidase Y/Usp8 (8, 9) and Grb-2 related protein (GADS), SH3 domain binding to SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) (10 -13). In the latter case, the interaction has a 10 -20 times higher affinity than interaction between SH3 domains and their øPXøP motifs. Other examples of non-canonical interactions include amphiphysin SH3 domain binding to dynamin (14), Eps8 SH3 domain binding to the PXXDY motif (15), and HPK1 SH3 domain binding to a RXXK motif (12). We recently identified a novel RKXXYXXY motif that is found in the T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) and that is recognized by the FYN-SH3 and ADAP-SH3c domains (16).In the immune system, SH3 domains are found in Src-related kinases and immune specific adaptor proteins (17, 18). Adaptors lack enzymatic activity and transcription binding domains, and instead are comprised of multiple binding domains and sites that facilitate protein-protein aggrega- Grb2, growth factor receptor-bound protein-2; Gads, Grb-2-related protein; SLP...

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Section: Discussionmentioning

confidence: 99%

Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein

Duke‐Cohan

Kang

Liu

et al. 2006

Journal of Biological Chemistry

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“…Then, after eliminating coefficients, (17) and (18) For the boundary between regions 2 and 3 (r = a 2 ), (19) (20) (21) and (22) Other boundaries are treated similarly. At r = a N (cavity wall), the boundary condition gives (23) 2.1.2.…”

Section: Methodsmentioning

confidence: 99%

“…Conductivities of all solutions were determined using a CDM83 conductivity meter (Radiometer, Copenhagen, Denmark) at T = 25 °C. The meter was calibrated using a 0.005 M KCl (718 ± 1 S/cm at T = 25 °C [18]). PADS, K 2 CO 3 , TEMPO, KCl, and Na 2 HPO 4 were purchased from Aldrich (Milwaukee, WI).…”

Section: Methodsmentioning

confidence: 99%

“…8). Increase of sample conductivity has a dramatic effect on NMR sensitivity, i.e., change of conductivity from = 2 S/cm (doubly distilled water) to = 22 mS/cm (disodium phosphate aqueous solution, concentration 200 mM) decreases sensitivity by a factor of 4 [18]. In EPR, this change of non-saturated sample conductivity decreases the signal intensity by only 5% (Fig.…”

Section: Non-saturated Aqueous Samplementioning

confidence: 99%

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Aqueous sample in an EPR cavity: sensitivity considerations

Nesmelov

Gopinath

Thomas

2004

Journal of Magnetic Resonance

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The radial mode matching (RMM) method has been used to calculate accurately the microwave field distribution of the TE 0 1 1 mode in a spherical EPR cavity containing a linear aqueous sample, in order to understand in detail the factors affecting sensitivity in EPR measurements at X band. Specific details of the experiment were included in the calculations, such as the cavity geometry, the presence of a quartz dewar, the size of the aqueous sample, and the sample's dielectric properties. From the field distribution, several key physical parameters were calculated, including cavity Q, filling factor, mean microwave magnetic field at the sample, and cavity efficiency parameter . The dependence of EPR signal intensity on sample diameter for a cylindrical aqueous sample was calculated and measured experimentally for non-saturated and half-saturated samples. The optimal aqueous sample diameter was determined for both cases. The impact of sample temperature, conductivity, and cavity Q on sensitivity of EPR is discussed.

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“…Such high salt concentrations present a problem for high-resolution structural techniques such as NMR. They introduce high dielectric constant and electrical conductivity, which, in turn, severely compromises the NMR probe performance and leads to unavoidable radiofrequency-induced heating detrimental to both the sample and the NMR probe (Kelly et al 2002;Stringer et al 2005). In the past, this has limited the investigation of the structure of group II introns under functional conditions by NMR.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme

Gumbs¹,

Padgett²,

Dayie³

2006

RNA

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We have reconstructed the group II intron from Pylaiella littoralis (PL) into a hydrolytic ribozyme, comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans that efficiently cleaves spliced exon substrates. Using a novel gelbased fluorescence assay and nuclear magnetic resonance (NMR) spectroscopy, we monitored the direct binding of D5 to D123, characterized the kinetics of the spliced exon hydrolysis reaction (which is mechanistically analogous to the reverse of the second catalytic step of splicing), and identified the binding surface of D123 on D5. This PL ribozyme acts as an RNA endonuclease even at low monovalent (100 mM KCl) and divalent ion concentrations (1-10 mM MgCl 2 ). This is in contrast to other group II intron ribozyme systems that require high levels of salt, making NMR analysis problematic. D5 binds tightly to D123 with a K d of 650 6 250 nM, a K m of ;300 nM, and a K cat of 0.02 min À1 under single turnover conditions. Within the ;160-kDa D123-D5 binary complex, site-specific binding to D123 leads to dramatic chemical shift perturbation of residues localized to the tetraloop and internal bulge within D5, suggesting a structural switch model for D5-assisted splicing. This minimal ribozyme thus recapitulates the essential features of the reverse of the second catalytic step and represents a wellbehaved system for ongoing high-resolution structural work to complement folding and catalytic functional studies.

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Low-Conductivity Buffers for High-Sensitivity NMR Measurements

Cited by 161 publications

References 24 publications

Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein

Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein

Aqueous sample in an EPR cavity: sensitivity considerations

Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme

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