Complete 13C resonance assignments of tryptophan in L-lysyl-L-tryptophyl-L-lysine by single-bond and multiple-bond correlated 1H13C two-dimensional NMR
✍ Scribed by B.-H. Oh; J. L. Markley
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1989
- Tongue
- English
- Weight
- 261 KB
- Volume
- 28
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
Recent application of two-dimensional (2D) 13C{'3C} double-quantum correlated ['3C{'3C}DQC] nmr spectroscopy to the study of uniformly labeled proteins has enabled extensive assignments of 13C resonances. '-3 In analyzing the 13C spin systems of tryptophan residues in flavodoxin and cytochrome k3 from Anubaena 7120,4 we found that the carbon assignments did not match the most recent ones in the literature for tryptophan in model peptides? Moreover, in reviewing the literature, we found conflicting and/or ambiguous assignments reported by several groups?-"
In order to settle the controversy concerning valid reference values for 13C chemical shifts of tryptophan in peptides and proteins, we carried out the nmr analysis of a tryptophan-containing tripeptide (L-lysyl-L-tryptophyl-L-lysine). The nmr methods used were one-dimensional (1D) 'H-nmr spectroscopy with single-frequency 'H decoupling, 2D 'H{13C} single-bond correlation [' H{13C}SBC]:2 and 'H{I3C} multiple-bond correlation [1H{'3C}MBC]13*14 spectroscopy. Heteronuclear 2D 'H-I3C chemical shift correlation nmr methods provide reliable resonance assignments of these nuclei and overcomes ambiguities associated with traditional I3C signal assignments that rely on empirical additivity rules or chemical shift comparisons with related m~lecules.'~ The 2D 'H{13C}SBC experiment provides one-bond connectivities of C-H groups and thus effectively gives the same information as a set of 1D 13C-nmr spectra with single-frequency proton decoupling. The 'H{'3C}MBC experiment yields long-range (2-3 bond) 'H-I3C coupling information that provides multiple connectivities between carbon and hydrogen atoms.
Methods
L-lysyl-L-tryptophyl-L-lysine and L-tryptophan were purchased from Sigma Chemical Co., and used without further purification. The tripeptide sample used for nmr spectroscopy was 50 mM L-lysyl-L-tryptophyl-L-lysine dissolved in 2.8 mL 'H,O containing 100 m M phosphate buffer at pH* 7.24 (pH reading uncorrected for deuterium isotope effect). The free amino acid sample was 28 mM L-tryptophan dissolved in dimethylsulfoxide (DMSO-d,). All nmr data were collected at 25°C on a Bruker AM-400 WB nmr spectrometer operating at a 'H frequency of 400.13 MHz and a I3C frequency of 100.6 MHz. The 'H{'3C}SBC and 'H(l3C}MBC spectra were collected with a Bruker 10-mm broad-band probe by using Bruker reverse electronics. The 'H{'3C}SBC spectrum was recorded without I3C decoupling during acquisition; the cross peaks appear as doublets separated by 'JcH . The delay time used was 3.12 ms to optimize for 160 Hz 'H-I3C couplings. For the 'H{13C}MBC *To whom correspondence should be addressed.