De novo design of native proteins: characterization of proteins intended to fold into antiparallel, rop-like, four-helix bundles.
The de novo design and characterization of a series of 51-residue helix-turn-helix peptides intended to dimerize into antiparallel four-stranded coiled coils is described. The sequence is based on a coiled coil heptad repeat Ncap-(Aa Zb Zc Ld Ze Zf Zg)3-turn- (Xa Zb Zc Ld Ze Zf Zg)3-Ccap-CONH2, where X is either Val or Ala. The overall topology was intended to be similar to that found in the Escherichia coli protein ROP. The design strategy included consideration of geometric complementarity of the packing of side chains within the hydrophobic core as well as the use of specific interfacial interactions, both of which were intended to favor the desired ROP-like topology. Additionally, the sequence was designed to destabilize potential alternative structures that might compete with the desired topology. The peptides (RLP-1, RLP-2, and RLP-3) assemble into stable alpha-helical dimers and exhibit the hallmarks of a native protein as judged by its spectroscopic properties, and the lack of binding to hydrophobic dyes. Also, the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence of the CD spectra and confirmed by differential scanning calorimetry (DSC). The values determined by the two methods are in excellent agreement and are in the range of those of naturally occurring proteins of this size. These results suggest that it is now possible to design native-like helical proteins that should serve as templates for the further design of functional proteins.
Betz
S F
SF
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104-6059, USA.
Liebman
P A
PA
DeGrado
W F
WF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli; D018257:Helix-Loop-Helix Motifs; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D016601:RNA-Binding Proteins / Q000737:chemistry*; D013816:Thermodynamics
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; C054243:Rop protein, ColE1 plasmid