Mutagenesis and Peptide Synthesis Protocol

All chemicals used were purchased from Sigma (St. Louis, MO) unless otherwise noted. NMR tubes were purchased from Shigemi. (Allison Park, PA). Nitrocefin was purchased from EMD Millipore (Billerica, MA). BL21 competent cells purchased from Agilent Technologies. Plasmids pDIMC8-c4h and pDIMC8-MBP that contain the c4 and malE genes respectively were previously described^{9 (link),10 (link)}. Plasmid pDIMC8-c4h was used as a template plasmid DNA to make linker variants using the PCR-based QuikChange mutagenesis procedure (Agilent Technologies). DNA sequencing of 2013 to 2061 bp regions containing the targeted codon confirmed mutant clones (Genewiz). The WA-(EAQA)₇ peptide (sequence: WAEAQAEAQAEAQAEAQAEAQAEAQAEAQA) was custom synthesized with 98% purity by Peptide 2.0. synthesis, and peptide ends were modified by N-terminal acetylation and C-terminal amidation.