Protein Engineering Protocols - Mycobacteriology research center

Design of Coiled Coil Structures 613. Notes1. Sequence of GCN4-p1: Ac-R MKQLEDK VEELLSK NYHLENE VARLKKLVGER-COOH. In boldface are the a and d residues that have been mutated in thestudies by Harbury et al. (16,17). Underlined is the core Asn16, which has beensubjected to many mutations.2. The designed heterodimeric Peptide Velcro (9) consists of the synthetic peptidesAcid-p1 (Ac-AQLEKE LQALEKE NAQLEWE LQALEKE LAQ-NH 2) andBase-p1 (Ac-AQLKKK LQALKKK NAQLKWK LQALKKK LAQ-NH 2). Inthis and the following notes, the individual heptads of the respective sequenceare separated by a space. The core Asn residue, which has been mutated asdescribed, e.g., in Subheading 2.1.2., item 4, is underlined.3. The sequence of the two antiparallel cysteine disulfide-bridged peptides 2H (Ac-KCEALEGK LEALEGK LEAAEGK LEALEGK LEALEG-NH 2and Ac-ELAELKGE LAELKGE AAELKGE LAELKGE LAECKG-NH 2) and 4H (Ac-KCEALEGK LEALEGK LEAAEGK LEALEGK LEALEG-NH 2and Ac-ELAELKGE LAELKGE LAEAKGE LAELKGE LAECKG-NH 2). Cysteines areshown in bold to indicate the point of covalent bonding between the two chains in2H and 4H, respectively, and the Ala residue that specifies the oligomerizationstate is underlined (20).4. Sequence of the coiled coil domain (amino acids 27–72) of the rat COMP protein(21): GDL APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT VMEC-DAC G. In the expressed fragment of rat COMP, Gly 27 was replaced by Met.5. Studies were based on the peptide A1 (MRGSHHHHHHGSMA SGDLENEYAQLERE VRSLEDE AAELEQK VSRLKNE IEDLAEI GDLNNTSGIRRPAAKLN). Incorporation of trifluoroleucine and hexafluoroleucine for Leucine (inboldface) was achieved by induction of gene expression in leucine-free culturemedia supplemented either with trifluoroleucine or with hexafluoroleucine (22,23).6. Kretsinger et al. based their study on the GCN4-p1 peptide as given in Note 2 withthe differences that the C-terminus was amidated, and that they reported a sequencewith an additional Ser between the Asp and Lys in the first full heptad (24).Because this addition would shift the heptad repeat, we presume that it is an errorin the figure. The underlined Asn (see Note 2) was subjected to exchange to Aspdap as well as monomethylated, dimethylated, and trimethylated analogs of dap.7. In this selection, heterodimers were selected from two designed coiled coillibraries: LibA: VAQL#E# VKTL#A# §YEL#S# VQRL#E# VAQL and LibB:VDEL#A# VDQL#D# §YAL#T# VAQL#K# VEKL, where # denotes an equimolarmixture of E, Q, K, and R, and § denotes an equimolar mixture of V and N(31). The sequences for the core a and d positions were taken from GCN4 (seeNote 2) and for the b, c, and f positions (underlined) were taken from the coiledcoil domains of c-Jun (IARLEEK VKTLKAQ NYELAST ANMLREQ VAQL)and c-Fos (TDTLQAE TDQLEDE KYALQTE IANLLKE KEKL), respectively.8. The sequence of the GCN4-pVL variant is Ac-R MKQLEDK VEE#LSK§YHLENE VARLKKL VGER, where the a and d positions are in boldface.