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. 2011 Jan;49(1):3-7.
doi: 10.1007/s10858-010-9464-2. Epub 2010 Dec 29.

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy

Mary Anne Refaei  1 , Al Combs, Douglas J Kojetin, John Cavanagh, Carol Caperelli, Mark Rance, Jennifer Sapitro, Pearl Tsang
Affiliations

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy

Mary Anne Refaei et al. J Biomol NMR. 2011 Jan.

Abstract

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is approximately 40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.

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Figures

Fig. 1
Fig. 1
Scheme of protein ligation by sortase A enzyme. The consensus sortase residues required at the end of the N-domain are shown (in red) followed by an ‘R’ group (typically His6, in black). Sortase-specific glycine residue(s) required at the N-terminus of the C-domain are in blue. Ligation of both domains results from formation of the new T-G bond (dashed line) that is catalyzed by sortase
Fig. 2
Fig. 2
Confirmation of successful sortase ligation by SDSPAGE and mass spectrometry. The SDSPAGE gel is shown on the left. The left lane corresponds to double mutant MecAmut (band ‘A’) while the remaining lanes correspond to the ligation mixture at the indicated times of incubation. The bands outlined in red were excised from the gel and subjected to tryptic digestion. On the right, the full length MecAmut sequence is shown with the tryptic peptides obtained from product band (‘B’) that were sequenced by MS/MS indicated in red. These sequenced fragments span both domains of full length MecAmut
Fig. 3
Fig. 3
2D 1H-15N HSQC spectra of fully 15N-labeled MecA double mutant (MecAmut) (top left) and N-terminally labeled 15N-MecAN-MecAC (MecAmut2G) protein conjugate (top right). Overlay of the two spectra is shown at bottom (black—fully labeled protein, red—N-terminal domain labeled). Spectra were acquired on a 600 MHz Varian Inova spectrometer at 303 K
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