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Introduction of a Polar Core Into the De Novo Designed Protein Top7

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Introduction of a Polar Core Into the De Novo Designed Protein Top7 Introduction of a polar core into the de novo designed protein Top7 Benjamin Basanta,1,2,3 Kui K. Chan,4 Patrick Barth,5,6,7 Tiffany King,8 Tobin R. Sosnick,8,9 James R. Hinshaw,10 Gaohua Liu,11,12 John K. Everett,11,12 Rong Xiao,11,12 Gaetano T. Montelione,11,12,13 and David Baker1,2,14* 1Department of Biochemistry, University of Washington, Seattle, Washington 98195 2Institute for Protein Design, University of Washington, Seattle, Washington 98195 3Graduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, Washington 98195, USA 4Enzyme Engineering, EnzymeWorks, California 92121 5Structural and Computational Biology and Molecular Biophysics Graduate Program, Baylor College of Medicine, Houston, Texas 77030 6Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030 7Department of Pharmacology Baylor College of Medicine, Houston, Texas 77030 8Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 9Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637 10Department of Chemistry, University of Chicago, Chicago, Illinois 60637 11Department of Molecular Biology and Biochemistry, Center of Advanced Biotechnology and Medicine, The State University of New Jersey, Piscataway, New Jersey 08854 12Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 13Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854 14Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195 Received 2 October 2015; Revised 4 February 2016; Accepted 8 February 2016 DOI: 10.1002/pro.2899 Published online 00 Month 2016 proteinscience.org Abstract: Design of polar interactions is a current challenge for protein design. The de novo designed protein Top7, like almost all designed proteins, has an entirely nonpolar core. Here we describe the replacing of a sizable fraction (5 residues) of this core with a designed polar hydrogen bond network. The polar core design is expressed at high levels in E. coli, has a folding free energy of 10 kcal/mol, and retains the multiphasic folding kinetics of the original Top7. The NMR structure of the design shows that conformations of three of the five residues, and the designed hydrogen bonds between them, are very close to those in the design model. The remaining two residues, which are more solvent exposed, sample a wide range of conformations in the NMR ensemble. These results show that hydrogen bond networks can be designed in protein cores, but also high- light challenges that need to be overcome when there is competition with solvent. Additional Supporting Information may be found in the online version of this article. Broader Audience Statement: Natural proteins have primarily hydrophobic cores and polar exteriors. With the long-term goal of mak- ing “inside out” proteins with polar cores, we explore the properties of a designed protein with a polar hydrogen bond network in its core Benjamin Basanta and Kui K. Chan contributed equally to this work Grant sponsor: US National Institutes of Health; Grant number: U54GM094597 (to G.T.M.); Grant sponsor: NIH; Grant number: GM055694 (to T.R.S); Grant sponsor: Howard Hughes Medical Institute. *Correspondence to: David Baker, University of Washington, Molecular Engineering and Sciences, Box 351655, Seattle, WA 98195- 1655. E-mail: [email protected] Published by Wiley-Blackwell. VC 2016 The Protein Society PROTEIN SCIENCE 2016 VOL 00:00—00 1 Keywords: protein design; hydrogen bonds; protein folding; protein NMR; protein core polar interactions Introduction mized by minimization in the Rosetta forcefield; the De novo protein design has advanced considerably lowest energy of the refined designs is shown in Fig- in recent years. New protein structures, functions, ure 1(C). and interactions have been successfully designed.1–5 In a first round of experiments, we attempted to Both the new structures and new interactions are express the two lowest energy designed “inside-out” stabilized primarily by nonpolar interactions; there proteins in Escherichia coli with the hope that they has been less success with hydrogen bonding and would form inclusion bodies that could then be solu- polar interaction networks. bilized in organic solvent. However, there was little The de novo designed Top7 protein has an or no expression of these rather unusual proteins. entirely hydrophobic core and is extremely stable.6 To evaluate the designed polar core independent of Characterization of the folding kinetics of Top7 such a drastic surface redesign, we replaced the sur- showed the process was considerably more complex face residues on the two lowest energy polar-core than that of most native proteins with the same Top7 designs with those of the original water-soluble size, with multiple distinct kinetic phases. Analysis design. As we anticipated that the polar core designs of fragments of Top7 showed that many had appreci- would be considerably destabilized, we decided to able structure in isolation. The high stability and follow the example of crambin, a small protein with low folding cooperativity of Top7 suggests that there a quite polar core,9 and incorporate a disulfide bond may have been more selection for folding cooperativ- into the design. We searched for pairs of positions at ity than stability during evolution.7 which substituted cysteines could form a disulfide With the long-range goal of designing an “inside bond by introducing cysteines at every residue and out” protein—with a polar core and a nonpolar exte- using the Rosetta all atom energy function to iden- rior—that could be stable in organic solvent, we tify residue pairs with low energy disulfide bonds. explored the replacement of a portion of the Top7 We identified three pairs of positions where disulfide hydrophobic core with a polar hydrogen bond net- bonds with good geometry could be introduced [Fig. work. The polar-core Top 7 (Top7_PC) is folded and 1(D), only the model for the best behaved protein is moderately stable, and structural characterization shown]. shows that the majority of the hydrogen bonds in We tested the designed disulfides initially in the the designed network are intact. context of the original nonpolar core Top7. The three disulfides were introduced by mutagenesis, and the Results proteins expressed and purified. One of the three With the goal of designing an “inside out” protein, introduced disulfides—between residues 12 and 45— we adapted the RosettaMembrane all-atom energy was formed quantitatively, as assessed using Ell- function which utilizes an implicit model of the lipid man’s reagent. Guanidine hydrochloride (GuHCl) surrounding the transmembrane portions of mem- denaturation experiments tracking far-UV circular brane proteins.8 We modified the energy function dichroism (CD) showed the introduced disulfide such that the protein environment was treated as a shifted the denaturation midpoint, Cm, of Top7, an uniform implicit apolar solvent instead of a hetero- already very stable protein, from 6.5M to 8M geneous lipid membrane. We first entirely rede- GuHCl (Top7_CC, Supporting Information Fig. 1). signed Top7 using the apolar potential, keeping the Because little or no unfolded baseline could be meas- backbone conformation fixed, and allowing all 20 ured, we could not determine the increase in folding amino acids at all 94 positions to assess whether free energy by fitting the full unfolding transition; networks of polar residues with low energy configu- instead we estimate from the shift in midpoint and rations could be designed in its core. The calcula- the m-value of Top7 that the disulfide increases sta- tions converged on very similar low energy designed bility by 3 kcal/mol. structures, with most core positions of the original We next introduced the 12 to 45 disulfide bond Top7 recapitulated except for five positions substi- into the protein having the designed core hydrogen tuted with polar residues forming a connected bond network, keeping the original Top7 surface res- hydrogen bond network with each residue stabilized idues at all other positions [Fig. 1(E)]. The resulting by two hydrogen bonds [Fig. 1(A,B)]. The polar net- protein “Top7_PC” expressed at high levels in E. coli work bridges several secondary structural elements and could be readily purified. The CD spectrum of in the Top7 core including the first and third beta Top7_PC was very similar to that of the wild-type strands and the C-terminal helix. The backbone and protein [Supporting Information Figs. 4 and 2(A) of side-chain dihedral angles of the designs were opti- Ref. 6]. Rotational correlation time (sc) estimates 2 PROTEINSCIENCE.ORG Polar Core in De Novo Designed Protein Top7 Figure 1. Top7_PC and Top7 models. Close up view comparing the core region of Top7 before (B) and after (A) hydrogen- bond network incorporation. (C) Hydrogen-bond network in the context of the whole structure of one of the initial “inverted” Top7 models. (D) Model of the disulfide-bonded variant of Top7. (E) Model of disulfide-bonded Top7 with core hydrogen-bond network. based on 15N NMR nuclear relaxation measure- teins are similar. Both proteins are stabilized ments (T1/T2) indicate that Top7_PC is predomi- entropically at low temperature but enthalpically at nantly monomeric in solution (Supporting
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