Total chemical synthesis of a 27 kDa TASP protein derived from the MscL ion channel of M. tuberculosis by ketoxime-forming ligation.
A 27-kDa TASP protein, T(5)Msc(103-151), that was derived from the cytoplasmic domain (amino acid residues 103-151) of the MscL ion channel of M. tuberculosis was synthesized by ketoxime-forming chemoselective ligation between a template molecule carrying five pyruvic acid groups, and linear channel peptides carrying one aminooxyacetic acid group. Ketoxime-forming ligation provided for highly efficient assembly of this large totally synthetic protein construct with yields >90% with modest excess (1.5x) of the aminooxy peptide. Formation of the desired TASP molecule was confirmed by SDS-PAGE analysis and MALDI mass spectrometry. The effect of template attachment on the structure of the peptides constituting the TASP was assessed by circular dichroism spectroscopy. Attachment of the peptides to the topological template induces predominantly helical secondary structure, whereas an analogous peptide that did not bear an aminooxy group, MscL(103-151), does not exhibit significant secondary structure at pH 7 and is found to be monomeric in concentrations up to 65 microM. This observation can be explained by entropic destabilization of the unfolded state of T(5)Msc(103-151) due to the attachment to the template and the resulting loss of degrees of freedom. Pyruvic acid-based ketoxime-forming chemoselective ligation may thus prove to be a useful tool for the assembly of large, non-native protein constructs and their biophysical study.
Kochendoerfer
Gerd G
GG
Gryphon Sciences, 250 East Grand Avenue, Suite 90, South San Francisco, California 94080, USA. Gkochendoerfer@gryphonsci.com
Tack
Janette M
JM
Cressman
Sonya
S
D001426:Bacterial Proteins / Q000138:chemical synthesis*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003593:Cytoplasm; D006863:Hydrogen-Ion Concentration; D008026:Ligation; D013058:Mass Spectrometry; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D010091:Oximes / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D013698:Templates, Genetic
D001426:Bacterial Proteins; D010091:Oximes