Development of the strategies for assembling multiple kinds of peptide segments would give new possibilities for the de novo design of functional proteins. We will introduce our approach for the selective assembly of helical peptide segments on a peptide template to give four-helix-bundle proteins comprising individual helices.1Abbreviations: A=alanine, E=glutamic acid, F=phenylalanine, G=glycine, I=isoleucine, K=lysine, L=leucine, M=methionine, N=asparagine, P=proline, Q=glutamine, R=arginine, S=serine, T=threonine, V=valine, W=tryptophan, Y=tyrosine, GABA=γ-aminobutyric acid, Fmoc=9-fluorenylmethyloxycarbonyl, Ac=acetyl, tBu=t-butyl, Boc=t-butyloxycarbonyl, Trt=trityl, MBzl=p-methoxybenzyl, Ad=adamantyl, Acm=acetamidomethyl, DICDI=diisopropylcarbodiimide, HOBt=1-hydroxybenzotriazole, NMM=N-methylmorpholine, TFA=trifluoroacetic acid, EDT=1,2-ethanedithiol, AgOTf=silver trifluoromethanesulfonate, DTT=dithiothreitol, AcOH=acetic acid, Tris=tris(hydroxymethyl)aminomethane, MOPS=3-(N-morpholino)propanesulfonic acid, DMF=dimethylformamide, MeOH=methanol, HPLC=high performance liquid chromatography, LSIMS=liquid secondary ion mass spectrometry, TOFMS=time of flight mass spectrometry, CD=circular dichroism.1