Protein Engineering Protocols - Mycobacteriology research center

64 Mason et al.25. Synthetic peptides with the following sequence were constructed: Ac-(K LEA-LEG) n-K-NH 2, with n = 1 to 5 (62) and compared with carboxamidomethylatedα-tropomyosin at cysteine 190 (CM-tropomyosin).26. A series of polypeptides containing 9, 12, 16, 19, 23, 26, 30, 33, and 35 amino acidresidues were designed with the sequence: Ac-E iealkae iealkae iealkae iealkaeieacka-NH 2for the 35-mer peptide (63). Shorter peptides were comprised of therespective number of amino acids counted from the C-terminus.27. The peptide Succ-DELERR IRELEAR IK-NH 2was used in this study (64), Succindicated the succinylated N-terminus.28. Investigated peptides were Lac 21: Ac-MKQLADS LMQLARQ VSRLESA-NH 2,Lac 28: Ac-LMQLARQ MKQLADS LMQLARQ VSRLESA-NH 2, and Lac 35:Ac-LMQLARQ LMQLARQ MKQLADS LMQLARQ VSRLESA-NH 2(65).29. Studies were based on the mutants of the E/K heterodimer comprised of theE-peptide with the sequence Ac-(E #§ALEK) n-NH 2and the K-peptide with thesequence Ac-(K #§ALKE) n-NH 2with n = 3 or 4. # indicates I or V; § indicatesA or S (67).30. The peptide sequence was Ac-Q CGALQKQ VGALQKQ VGALQKQ VGALQKQVGALQK-NH 2. Positions 1, 6, 15, 20, and 34 that were mutated to Gln are underlined(73).31. This study worked with recombinant GCN4-pMSE peptide (MS VKELEDKVEELLSK NYHLENE VARLKKL VGER). The capping motif is in boldface andmutations compared with GCN4-p1 (see Note 2) are underlined. Other peptidesfrom this study were GCN4-pSE, which lacked the initiator methionine, andGCN4-pAA, in which the Ser and Glu of GCN4-pSE were mutated to Ala (77).Stabilities of GCN4-pAA were comparable to GCN4-p1, whereas the GCN4-pSEand GCN4-pMSE variants were stabilized by 0.5 kcal/mol and 1.2 kcal/mol,respectively, relative to GCN4-pAA. Thus, the hydrophobic contribution of theterminal Met residue is 0.7 kcal/mol.32. Heterodimeric coiled coils, denoted GABH, for GCN4 (see Note 2) Acid/BaseHeterodimer were designed with the acidic sequence A (Ac-E VKQLEAEVEE#ESE #WHLENE VARLEKE NAECEA-NH 2) and the basic sequence B(Ac-K VKQLKAK VEE#KSK #WHLKNK VARLKKK NAECKA-NH 2) (98).Positions d12 and a16 (#) were mutated to Val, Ile, and Leu, respectively, to yieldthe peptides A LL,A IV, and A LI, and B LLand B LV.33. Designed sequences were dimeric RH2 (Ac-AE IEQLKKE§AYL IKKLKAEKLAEIKKLKQEKA-NH 2), trimeric RH3 (Ac-AE #EQ#KKEIAYL #KK#KAEILAE#KK#KQEIA-NH 2), and tetrameric RH4 (Ac-AE LEQ#KKEIAYL LKK#KAEIL AELKK#KQEIA-NH 2) (99). The hydrophobic residues (a, d, and h) of the undecatadrepeat (a to k) are marked in boldface; § indicates norvaline; and # indicatesalloisoleucine residues.AcknowledgmentThis work was funded in the Emmy Noether program of the DeutscheForschungsgemeinschaft (grant Ar 373).