Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution

Kainosho, Masatsune; Tsuji, Takashi

doi:10.1021/bi00267a036

Cited by 171 publications

(121 citation statements)

References 25 publications

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“…Use of REDOR filtering for larger proteins will require incorporation of specific 13 C and 15 N labels in an expression protocol [2,41,[62][63][64]. A single carbonyl site can be probed using the carboxylic 13 C-labeled amino acid of the residue of interest and the 15 N-labeled amino acid of the next residue in the sequence.…”

Section: Future Applicationsmentioning

confidence: 99%

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Yang

Parkanzky

Bodner

et al. 2002

Journal of Magnetic Resonance

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Clean MAS observation of 13 C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13 C-15 N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13 C carbonyl and its directly bonded 15 N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic-and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U-13 C, 15 N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13 C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.

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Section: Future Applicationsmentioning

confidence: 99%

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Yang

Parkanzky

Bodner

et al. 2002

Journal of Magnetic Resonance

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“…The major disadvantages in application of NMR spectroscopy are that of sensitivity and limitation in high-molecular mass systems. In order to overcome respectively these two obstacles, methods of in vitro or in vivo isotopic labeling with specific magnetically active nuclear spins (principally 13 C and 15 N) [2,3] and new pulse sequences to specifically reduce losses from transverse relaxation mechanisms [4][5][6] were developed along the years. These methods, established primarily to proteins and nucleic acids [4][5][6][7][8][9], are now being used to study carbohydrates and glycoconjugates as well [10,11].…”

Section: The Essential High-throughput Nmr Methods Exploited In Glycomentioning

confidence: 99%

“…Scalar coupling are usually easily seen by splitting resonances on 1D NMR spectra, but very often, J-resolved NMR experiments must be undertaken for obtaining such values. 3 JH-H can primarily serve as diagnostic for the ring structural conformers of certain glycans. The usefulness of scalar coupling constants can be used by the rule that -anomers usually exhibit larger 3 JH1-H2 values than -1 H1-anomers (Table 5, and Figure 2B), which thus help structural assignment.…”

Section: Scalar Couplings In Conformational Analysis Of Sugar Ringsmentioning

confidence: 99%

Unravelling Glycobiology by NMR Spectroscopy

Pomin¹

2012

Glycosylation

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“…The method utilizes selective 13 C labeling and direct 13 C signal detection (29,30,31). The approach takes advantage of isotope effects from the neighboring amide hydrogens on the carbonyl 13 C chemical shift ( , in which the first and second letters in the brackets indicate the isotopes occupying the γ-and β-sites, respectively.…”

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

Carbon-13 NMR Method for the Detection of Correlated Hydrogen Exchange at Adjacent Backbone Peptide Amides and Its Application to Hydrogen Exchange in Five Antiparallel β Strands within the Hydrophobic Core of Streptomyces Subtilisin Inhibitor (SSI)

2005

Self Cite

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A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the 13 C NMR signals from peptide carbonyls. The line shape of the carbonyl 13 C i signal is influenced by differential H/D occupancy at the two adjacent amides: the H N i+1 (β-site) and the H N i (γ-site). At a carbon frequency of 75.4 MHz, the H→D isotope shifts on the 13 C signal are about 5-7 Hz for exchange at the β-site and 2 Hz or less for exchange at the γ-site. Since the effects at the two sites are additive, the time-dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites, but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k c ) and degree of correlated exchange (X βγ ) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective 13 C′ labeling at all valyl residues and one with selective 13 C′ labeling at all leucyl residues. This permitted the direct observation by one-dimensional 13 C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i+1 positions. The residues investigated were located in an α-helix and in a five-stranded antiparallel β-sheet. Samples of the two labeled proteins were prepared at various pH*values, and 13 C NMR spectra were collected at 50 °C prior to and at various times subsequent to transferring the sample from H 2 O to 2 H 2 O. Most of the slowly exchanging amides studied were intramolecular hydrogen bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding, but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k β and k γ ) were proportional to [OH − ], and the * To whom correspondence should be addressed. Telephone: +81-426-77-2544+81-426-77- . Fax: +81-426-77-2544 NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 27. Published in final edited form as:Biochemistry. . This enabled us to extract the pH-independent exchange rates (k β°, k γ°, and k c°) . In all cases in which correlated exchange could be measured, the observed sigmoidal pH-dependence of X βγ could be replicated roughly from the derived pH-independent rates. KeywordsStreptomyces subtilisin inhibitor; SSI; carbonyl 13 C NMR; stable isotope assisted NMR; H/D exchange; amide hydrogen; isotope shift; β-shift; γ-shift; correlated hydrogen exchangeThe internal fluctuations of protein molecules are deeply related to their structure and stability and contribute to their functions. Protein fluctuations have been ...

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Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution

Cited by 171 publications

References 25 publications

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Unravelling Glycobiology by NMR Spectroscopy

Carbon-13 NMR Method for the Detection of Correlated Hydrogen Exchange at Adjacent Backbone Peptide Amides and Its Application to Hydrogen Exchange in Five Antiparallel β Strands within the Hydrophobic Core of Streptomyces Subtilisin Inhibitor (SSI)

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