De NoVo Design of Native Proteins: Characterization of Proteins Intended To Fold into Antiparallel, Rop-like, Four-Helix Bundles ² Stephen F. Betz, ‡ Paul A. Liebman, and William F. DeGrado* The Johnson Research Foundation, Department of Biochemistry and Biophysics, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104-6059 ReceiVed July 12, 1996; ReVised Manuscript ReceiVed October 22, 1996 X ABSTRACT: 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 N cap -(A a Z b Z c L d Z e Z f Z g ) 3 -turn- (X a Z b Z c L d Z e Z f Z g ) 3 -C cap -CONH 2 , 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 R-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. Significant progress has been achieved in the design of proteins from first principles (Nautiyal et al., 1995; Olofsson et al., 1995; Raleigh et al., 1995; Betz & DeGrado, 1996). Recently, we described a series of fundamental principles for the hierarchic de noVo design of R-helical bundles (Betz & DeGrado, 1996). Three principles guide the design process and include (i) the use of complementary packing within the hydrophobic core, (ii) the use of specific interfacial interactions to promote interhelical association, and (iii) the use of both core and interfacial interactions to destabilize potential alternate conformations. Here, we describe the design and characterization of helix-turn-helix peptides designed to dimerize into a four- helix bundle that mimics the topology found in the Escheri- chia coli protein ROP (Banner et al., 1987). ROP is composed of two 63-residue peptides that form an antiparallel four-stranded coiled coil in which the two connecting loops are on opposite sides of the structure (Figure 1). The proteins described here are designed to be an abstraction of that structure and have the sequence N cap -(A a Z b Z c L d Z e Z f Z g ) 3 - turn-(X a Z b Z c L d Z e Z f Z g ) 3 -C cap -CONH 2 , where the a-g sub- scripts refer to the canonical coiled coil heptad repeat position (Cohen & Parry, 1990) and X is either alanine or valine (Figure 2). Three different peptides, RLP-1, -2, and -3 (where the number refers to the number of valine residues in the second helix), were synthesized and shown to form dimers in solution, consistent with their design. The behavior of these proteins is compared to that of naturally occurring proteins and other designed R-helical bundles. MATERIALS AND METHODS Generation of R-Helical Bundles. The design of the RLP series of proteins followed the procedures described previ- ² S.F.B. and W.F.D. were supported in part by the MRSEC Program of the National Science Foundation under Award DMR96-32598 and by National Institutes of Health Grant GM54616-01. P.A.L. was supported by National Institutes of Health Grants EY00012 and EY01583. * To whom correspondence should be addressed. Phone: 215-898- 4590. Fax: 215-573-7229. E-mail: wdegrado@mail.med.upenn.edu. ‡ Current address: Abbott Laboratories, 247G, 100 Abbott Park Road, Abbott Park, IL 60064. X Abstract published in AdVance ACS Abstracts, January 15, 1997. FIGURE 1: Molscript diagram (Kraulis, 1991) showing the structure and helix nomenclature of the E. coli protein ROP (Banner et al., 1987). 2450 Biochemistry 1997, 36, 2450-2458 S0006-2960(96)01704-7 CCC: $14.00 © 1997 American Chemical Society