Protein Engineering Protocols - Mycobacteriology research center

62 Mason et al.Position 12(d), indicated by #, is either a Leu or a polar residue (N, Q, S, or T),and position 16(a), indicated by §, is either a Val or a polar residue (25).9. Both studies used a disulfide-bridged coiled coil based on the model sequenceVGALKKE, with some modification to avoid intrachain and interchain charge–charge interactions with the site of substitution (X) and to adjust the overallcharge. The sequences were Ac-CGGE VGALKAQ VGALQAQ XGALQKEVGALKKE VGALKK-NH 2(33) and Ac-CGGE VGALKAE VGALKAQ IGAX-QKQ IGALQKE VGALKK-NH 2(32), respectively.10. Ji et al. used a recombinant model of the simian immunodeficiency virus gp41core, designated N36(L6)C34, where the amino-terminal helices (N36) form acentral, trimeric coiled coil, while the carboxyl-terminal helices (C34) pack in anantiparallel orientation into hydrophobic grooves on the surface of this coiled coiltrimer. N36 and C34 are separated by a short linker (L6; ref. 34). Mutations ofpolar core residues (in boldface) to Ile were made in the N36 domain: AGIVQQQQQLLDV VKRQQEL LRLTVWG TKNLQTR VT. The Q → I mutation thatformed insoluble aggregates is underlined.11. The sequence of the parent peptide, Lac21 is: Ac-MKQLADS LMQLARQ VSR-LESA-NH 2(see also Note 28). Underlined residues were mutated to E or K to resultin the peptides Lac21E and Lac21K, respectively, which formed a heterotetramer (35).12. Sequence of APC-55: AAAS YDQLLKQ VEALKME NSNLRQE LEDNSNHLTKLETE ASNMKEV LKQLQGS I and of anti-APCp1: MAAK GDQLKKEVEALEYE NSNLRKK LEDHKKK LTKLKTE ISNAKKM LKQLYAS I (36). Corechanges in anti-APCp1 compared with APC-55 are marked in boldface; changes atthe e and g positions are underlined; and changes to increase stability, to increase thenet charge to facilitate purification, and to add chromophores are marked in italics.13. The three peptides were T 9: Ac-R MKQLEKK XEELLSK AQQLEKE AAQLKKLVG-NH 2,T 16: Ac-R MKQLEKK AEELLSK XQQLEKE AAQLKKL VG-NH 2,T 23: Ac-R MKQLEKK AEELLSK AQQLEKE XAQLKKL VG-NH 2(37).Residues that were different in all three peptides are marked in boldface, X denotesthe cyclohexylalanine residue.14. Sequences were based on Acid-pLL and Base-pLL, which are identical to Acid-p1and Base-p1 (see Note 1) but with the core Asn mutated to Leu (see also Subheading2.1.2., item 4). Two L → K mutations (in boldface) were made to Base-pLL to yieldBase-pK: Ac-AQLKKK LQALKKK KAQLKWK KQALKKK LAQ-NH 2(38).15. Studies were based on an N-terminally capped variant of GCN4 (see Note 31;ref. 7) with the Asn16 shifted by one heptad level to position 9, yielding the peptidep-CAP: S VKELEDK NEELLSX XYHXXNE VARLKKL VGER. Changes comparedwith GCN4-p1 (see Note 2) are marked in boldface. X denotes positionsallowed to vary in the design calculations (39).16. Studies were based on the designed homodimeric coiled coil EK: Ac-KCGALEKK LGALEKK AGALEKK LGALEKK LGALEK-NH 2. Three mutantcoiled coils were made in which:a. Five Glu residues at e positions in EK (underlined) were mutated to Gln residues(peptide QK).