MUSIC in Triple-Resonance Experiments: Amino Acid Type-Selective 1H–15N Correlations

Schubert, Mario; Smalla, Maika; Schmieder, Peter; Oschkinat, Hartmut

doi:10.1006/jmre.1999.1881

Cited by 83 publications

(58 citation statements)

References 50 publications

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“…The spectra were processed with NMRPipe (31) and analyzed using Cara (32). For further diminishing of side chain ambiguity, three-dimensional CC(CO)NH (33,34) and several two-dimensional 1 H-15 N MUSIC (multiplicity selective in-phase coherence transfer) (35)(36)(37) experiments were conducted on the Bruker BioSpin AV600 spectrometer. The 1 H and 13 C chemical shifts were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid as an internal standard.…”

Section: Prokaryotic Expression and Purification Of Recombinantmentioning

confidence: 99%

Human Protein N-terminal Acetyltransferase hNaa50p (hNAT5/hSAN) Follows Ordered Sequential Catalytic Mechanism

Evjenth

Brenner

Thompson

et al. 2012

Journal of Biological Chemistry

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Section: Prokaryotic Expression and Purification Of Recombinantmentioning

confidence: 99%

Human Protein N-terminal Acetyltransferase hNaa50p (hNAT5/hSAN) Follows Ordered Sequential Catalytic Mechanism

Evjenth

Brenner

Thompson

et al. 2012

Journal of Biological Chemistry

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“…All NMR experiments were performed at 298 K using a sample containing 0.9 mM U- 13 (36), and residue selective MUSIC experiments specific for glycine, serine, and valine-isoleucine-alanine residues (37,38).…”

Section: Methodsmentioning

confidence: 99%

“…6). For instance, mutation Q40P (24) will destabilize helix I, due to the resulting absence of the hydrogen bonds with Leu 37 . Analogously, the mutation L62R (20) may reduce the stability of the whole PAI subdomain due to the crucial hydrophobic contacts with several other residues in the N-terminal HTH motif.…”

Section: Tablementioning

confidence: 99%

The Solution Structure of DNA-free Pax-8 Paired Box Domain Accounts for Redox Regulation of Transcriptional Activity in the Pax Protein Family

Codutti

Ingen

Vascotto

et al. 2008

Journal of Biological Chemistry

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Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redoxbased mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys 45 and Cys 57 ). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys 45 and Cys 57 and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors.Pax-8 is an important eukaryotic transcription factor that is responsible, during embryogenesis, for the differentiation of several organs such as kidney, thyroid, and neural tube. Pax-8 is also essential in the adult organism, where it activates thyroid hormone production. During development, Pax-8 functionality is regulated by alternative splicing, generating isoforms that exhibit different transactivation properties but maintain unaltered the DNA binding domain (1, 2). In mature thyroid follicular cells, the complete Pax-8 splicing isoform regulates the expression of thyroglobulin, thyroperoxidase (3, 4), sodium/ iodide symporter, and thyrotropin receptor genes (5-7). Pax-8 activity is co-regulated by thyroid transcription factor-1, thyroid transcription factor-2, and Hex, indicating an active role in the early commitment and differentiation of thyrocytes (8, 9). The Pax family shares a bipartite functionality consisting of a N-terminal binding region and a C-terminal transactivation region.The N-terminal region is usually comprised of three domains, namely a Paired Box (Prd) domain, a conserved octapeptide, and a further homeodomain (see Fig. 1). Differences in conservation and functionality in these domains have been used as evolution markers for the Pax family (10), which has been subdivided into four groups according to the sequence homology in the N-terminal region. In particular, the N-terminal region of Pax-8 is composed by all three domains but presents an incomplete and inactive homeodomain. The Prd domain consists of a well conserved 128-residue-long region, formed by two distinct subdomains known in literature as PAI (N-terminal) and RED (C-terminal) (11).DNA binding studies have demonstrated that the two subdomains of the Pax-8 Prd domain bind DNA independently (12), are both required f...

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“…Moreover, information about the amino acid type of a pseudoresidue can be included into MARS assignment. This information can come from a variety of sources, such as amino acid specific labeling (Lemaster and Richards, 1985;Ou et al, 2001), backbone resonance experiments that select only signals from specific amino acids (Dotsch et al, 1996;Schubert et al, 1999) or amide peaks in a (H)C(CO)NH-TOCSY spectrum Figure 2. Empirically optimized scheme for avoiding errors due to inaccuracies in predicted chemical shifts when mapping pseudoresidue segments to the protein sequence.…”

Section: Input Datamentioning

confidence: 99%

Mars – robust automatic backbone assignment of proteins

2004

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MARS a program for robust automatic backbone assignment of 13 C/ 15 N labeled proteins is presented. MARS does not require tight thresholds for establishing sequential connectivity or detailed adjustment of these thresholds and it can work with a wide variety of NMR experiments. Using only 13 C α / 13 C β connectivity information, MARS allows automatic, error-free assignment of 96% of the 370-residue maltose-binding protein. MARS can successfully be used when data are missing for a substantial portion of residues or for proteins with very high chemical shift degeneracy such as partially or fully unfolded proteins. Other sources of information, such as residue specific information or known assignments from a homologues protein, can be included into the assignment process. MARS exports its result in SPARKY format. This allows visual validation and integration of automated and manual assignment.

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MUSIC in Triple-Resonance Experiments: Amino Acid Type-Selective 1H–15N Correlations

Cited by 83 publications

References 50 publications

Human Protein N-terminal Acetyltransferase hNaa50p (hNAT5/hSAN) Follows Ordered Sequential Catalytic Mechanism

Human Protein N-terminal Acetyltransferase hNaa50p (hNAT5/hSAN) Follows Ordered Sequential Catalytic Mechanism

The Solution Structure of DNA-free Pax-8 Paired Box Domain Accounts for Redox Regulation of Transcriptional Activity in the Pax Protein Family

Mars – robust automatic backbone assignment of proteins

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