Networks of electrostatic and hydrophobic interactionsmodulate the complex folding free energy surface ofa designed βα proteinSujit Basaka, R. Paul Nobregaa,1, Davide Tavellaa, Laura M. Deveaua,1, Nobuyasu Kogab, Rie Tatsumi-Kogab,David Bakerc,d, Francesca Massia,2, and C. Robert Matthewsa,2

aDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; bExploratory ResearchCenter on Life and Living Systems, Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, 444-8585 Aichi, Japan; cHoward HughesMedical Institute, University of Washington, Seattle, WA 98105; and dDepartment of Biochemistry, University of Washington, Seattle, WA 98105

Edited by Martin Gruebele, University of Illinois at Urbana–Champaign, Urbana, IL, and approved February 14, 2019 (received for review November 1, 2018)

The successful de novo design of proteins can provide insights intothe physical chemical basis of stability, the role of evolution inconstraining amino acid sequences, and the production of custom-izable platforms for engineering applications. Previous guanidinehydrochloride (GdnHCl; an ionic denaturant) experiments of adesigned, naturally occurring βα fold, Di-III_14, revealed a cooper-ative, two-state unfolding transition and a modest stability.Continuous-flow mixing experiments in our laboratory revealeda simple two-state reaction in the microsecond to millisecond timerange and consistent with the thermodynamic results. In strikingcontrast, the protein remains folded up to 9.25 M in urea, a neutraldenaturant, and hydrogen exchange (HDX) NMR analysis in waterrevealed the presence of numerous high-energy states that inter-convert on a time scale greater than seconds. The complex protec-tion pattern for HDX corresponds closely with a pair of electrostaticnetworks on the surface and an extensive network of hydrophobicside chains in the interior of the protein. Mutational analysis showedthat electrostatic and hydrophobic networks contribute to the resis-tance to urea denaturation for the WT protein; remarkably, singlecharge reversals on the protein surface restore the expected ureasensitivity. The roughness of the energy surface reflects the denselypacked hydrophobic core; the removal of only two methyl groupseliminates the high-energy states and creates a smooth surface. Thedesign of a very stable βα fold containing electrostatic and hydro-phobic networks has created a complex energy surface rarely ob-served in natural proteins.

folding free energy surface | de novo-designed proteins | hydrogenexchange | partially folded states

The sequence space occupied by natural proteins is a smallfraction of the total space accessible to a heteropolymer with

20 monomeric units (1, 2). Evolved sequences are constrained bythe requirements to support ∼1,000 natural motifs (3) and tofold to a thermodynamically stable structure within secondswhile maintaining biological function and avoiding aggregation.One might wonder if de novo-designed sequences not limited byhistory and biological constraints are capable of folding to unique,stable structures on a smooth energy surface typically observed fornatural proteins. In the late 1980s, Regan and DeGrado (4) sys-tematically aimed to synthesize a four-helix bundle protein thatadopts stable folded structure in aqueous solution. Later, Harburyet al. (5) designed an unnatural right-handed tetrameric coiledcoil based on design principles extracted from natural left-handeddimeric coiled coils. Subsequently, Kuhlman et al. (6) designedthe sequence of a βα protein, Top7, also with a topology not foundin nature. Top7 was also capable of folding to a well-definedstructure (6), but its kinetic folding mechanism was complex andplagued by aggregation (7). Whether these unintended propertiesare a consequence of the novel topology or inherent to the se-quence remains to be determined.

The Baker group has gone on to design small βα proteins (8,9), multisubunit proteins (10, 11), membrane proteins (12), andeven virus-like capsids (13) that achieve the desired structureswith substantial stability. The overall design approach is tomaximize favorable side-chain interactions and minimize back-bone strain in the target folded state, and partially folded statesand possible folding pathways are not considered. Koga et al. (8)created sequences that adopted five different arrangements ofα-helices and β-strands with the desired structures and stabilities:the Ferredoxin-like fold, the Rossmann 2 × 2 fold, the IF3-likefold, the P-loop 2 × 2 fold, and the Rossmann 3 × 1 fold. Eachcontains a four-stranded β-sheet with a nonsequential strandorder. The Rossmann 2 × 2 fold, the P-loop 2 × 2 fold, and theRossmann 3 × 1 fold flank the β-sheet with α-helices on bothfaces, whereas the Ferredoxin-like fold and the IF3-like foldhave α-helices on only one face. Although these designed βαproteins achieved the desired structures and stability, their ki-netic folding mechanisms, in which the complexities in thefolding free-energy surface of Top7 were encountered, werenot explored.A thermodynamic and kinetic analysis of the folding mechanism

of one of the designed proteins, Di-III_14, a candidate from the

Significance

Natural protein sequences have evolved to fold efficiently totheir functional forms, traversing a relatively smooth energysurface with minimal frustration. The optimization of stabilityin de novo-designed proteins, without regard to dynamicalprocesses required for function, can have unintended conse-quences on energy surfaces. Electrostatic and hydrophobicnetworks of side chains for Di-III_14, a small βα protein with anatural topology, create a rough energy surface whose high-energy states reflect the progressive unlocking of a tightlypacked interior. Unbiased explorations of sequence space canreveal complex properties not seen in natural proteins, expandingour understanding of the relationships among sequence, struc-ture, folding, and function of these ubiquitous biopolymers.

Author contributions: S.B., F.M., and C.R.M. designed research; S.B., R.P.N., D.T., andL.M.D. performed research; S.B., R.P.N., D.T., L.M.D., N.K., R.T.-K., and D.B. contributednew reagents/analytic tools; S.B., R.P.N., D.T., and L.M.D. analyzed data; and S.B., F.M.,and C.R.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1Present address: Department of Protein Analytics and Antibody Engineering, Adimab,Lebanon, NH 03766.

2To whom correspondence may be addressed. Email: francesca.massi@umassmed.edu orc.robert.matthews@umassmed.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1818744116/-/DCSupplemental.

Published online March 15, 2019.

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IF3-like fold family and containing 74 aa, revealed a simpletransition on a smooth energy surface from a guanidine hydro-chloride (GdnHCl)-denatured state to the native state that occursin a few dozen microseconds upon dilution into buffer. Surpris-ingly, hydrogen exchange (HDX) NMR analysis in water revealedthe presence of numerous slowly interchanging high-energy statesthat require days for the complete exchange of main-chain amidehydrogens (NHs) with solvent. The observed protection patternsare closely associated with a pair of electrostatic networks on thesurface and a complementary network of nonpolar interactions inthe hydrophobic core. Evidently, there are unexplored regions ofsequence space for a natural fold that have complex free-energysurfaces not revealed by a classical chemical denaturation analysis.These observations have implications for protein design.

ResultsChemical Denaturation of Di-III_14 with GdnHCl Reveals a SmoothEnergy Surface. Di-III_14 was reversibly denatured with GdnHCl,a chemical denaturant that typically induces a self-avoiding ran-dom coil structure for globular proteins (14). In excellent agree-ment with a previous measurement, the CD data were welldescribed by simple two-state process (N ⇌ U) and yielded apredicted stability in water of 5.54 ± 0.18 kcal·mol−1 (Fig. 1A);compare with 5.6 kcal·mol−1 from the Baker laboratory (8). Furthersupport for the two-state model was obtained by extracting anearly identical stability from the unfolding transition monitored

by the fluorescence lifetime of the single tryptophan at position 63in β3 (SI Appendix, Fig. S1 and Table S1).Stopped-flow (SF) total intensity and continuous-flow (CF)

time-resolved fluorescence were employed to monitor the dy-namic unfolding and refolding reactions of Di-III_14 in GdnHClfor a time range from microseconds to seconds. A chevron plotof these data, i.e., the log of the relaxation time, τ, as a functionof denaturant concentration, is shown in Fig. 1B. The unfoldingand refolding reactions display simple exponential behavior witha maximum relaxation time of ∼20 ms near 2.4 M GdnHCl, themidpoint, Cm, of the equilibrium unfolding reaction. Unfoldingand refolding reactions accelerate exponentially above and belowthis denaturant concentration, as expected for a two-state mech-anism. Fitting the chevron to a two-state model (15), the unfoldingand refolding relaxation times in the absence of denaturant wereobtained by linear extrapolation and found to be 479 ms and 48 μs,respectively. These relaxation times correspond to rate constantsfor unfolding and refolding, ku and kr, of 2.09 s

−1 and 2.08 × 104 s−1,respectively [τ = (1/k)]. The stability calculated from the kineticdata for the transition over a single barrier from the N to the Ustate, 5.44 ± 0.12 kcal·mol−1 (Fig. 1B and SI Appendix, Table S1),is in excellent agreement with that measured from equilibriumexperiments. The concordant thermodynamic and kinetic resultsprovide further support for a simple two-state folding mecha-nism for Di-III_14 when denatured by GdnHCl.

HDX Reveals a Rough Energy Surface for Di-III_14 in Water. HDX ofNHs with water provides an independent assessment of thefolding free-energy surface of Di-III_14. There are two limitingscenarios. (i) First, the NHs exchange with solvent deuterium viaan EX1 mechanism in which refolding from the exchange com-petent state to the protected state is slower than exchange of theexposed NH with solvent deuterium. In this case, exchange islimited by the rate constant of unfolding (16). (ii) Alternatively,the NHs exchange via an EX2 mechanism, whereby the rateconstant of refolding is faster than the rate constant of exchange.In this case, exchange is modulated by the free energy differencebetween the protected and unprotected states. The definitive testof the mechanism and the correct interpretation of the HDXresults involves measurement of the exchange at different pHvalues. The rate constants for exchange are independent of pHfor the EX1 mechanism and increase by 10-fold for an increasein pH of 1 unit for the EX2 mechanism (17).We found that the exchange process requires days for comple-

tion (SI Appendix, Fig. S2) and is independent of pH between pH6.0, pH 6.5, and pH 7.4 for 29 measurably protected NHs, con-sistent with an EX1 process (Fig. 2A). The exchange of these NHsis limited by access to numerous exchange-competent states and isa highly noncooperative process. EX1 exchange kinetics for Di-III_14 require that the rate constant for refolding to the state of-fering protection against exchange be at least 10-fold slower thanthe intrinsic rate constant for exchange of the exposed NH. Basedon published intrinsic exchange rate constants (18), the refoldingrate constants must be <0.1 s−1 and the lifetimes of the exchangecompetent states >10 s. This behavior is in distinct contrast to thesmooth energy surface found for GdnHCl denaturation, in whichthe 2.08 × 104-s−1 refolding rate constant in water leads to theexpectation of an EX2 mechanism for exchange.

Urea Denaturation as a Test of the Role of Electrostatic Interactions inDefining the Energy Surface of Di-III_14. The contrasting views ofthe folding free-energy surface for Di-III_14 by GdnHCl denatur-ation and HDX in water motivated the use of an unchargedchemical denaturant, urea. Di-III_14 has an unnaturally high butbalanced content of cationic and anionic side chains, 35%, thatform multiple salt bridges on the surface. We tested whether theelectrostatic screening by the high salt concentrations with GdnHClis responsible for differing views of the energy surface by employingurea to denature Di-III_14. Generally, GdnHCl and urea providecomparable estimates of stability for natural proteins (19).

A C

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Fig. 1. Thermodynamic and kinetic analyses of Di-III_14. (A) GdnHCl de-naturation melts of the His6 (○, ●) and ΔHis6 (◊, ♦) constructs at pH 6.0(open symbols) and pH 7.4 (closed symbols) at 25 °C. The data were globallyfit to a two-state model (solid line). (B) A semilog plot of the relaxationtimes, τ, acquired from SF and CF kinetic folding experiments against finaldenaturant concentration; the symbols are identified in A. The solid line il-lustrates the fit of the kinetic data to a two-state model. The buffer in allexperiments contained 100 mM NaCl, 5.6 mM Na2HPO4, and 1.1 mM KH2PO4.(C) Urea titration of Di-III_14 in the absence of NaCl (○) and in the presenceof 2.5 M NaCl (●) monitored by the ellipticity at 222 nm for pH 7.4 and 25 °C.The expected urea titration curve for a stability of 5.54 kcal·mol−1 and anm-value of 1.2 kcal·mol−1·M−1 (dashed and dotted line). The fit of the partialtitration curve in the presence of 2.5 M NaCl (dotted line) yields a predictedstability, ΔG°, of 11.4 kcal·mol−1. A minimal estimate of the predicted sta-bility in water, 14 kcal·mol−1 (dashed line), reflects the stable native baselineup to 9.25 M urea. The solid black line indicates the GdnHCl denaturationmelt of Di-III_14. (D) Urea melt of Di_III-14 variants E32R (■, solid line), R69E(◊, dotted line), I70A (♦, dashed dotted line), V31A (hexagons, dashed line),and WT (●) measured by CD spectroscopy at 222 nm. The transition curveswere measured at 4 μM protein in 5.6 mM Na2HPO4, 1.1 mM KH2PO4, and100 mM NaCl at pH 7.4 and 25 °C. All curves were fitted to a two-state modelas described in the footnote to SI Appendix, Table S4.

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We were surprised to find that the protein remains folded upto the solubility limit of urea (Fig. 1C), 9.25 M. Based on thethermodynamic parameters for the GdnHCl melt, ΔG =5.54 kcal/mol, and m = 2.4 kcal/mol/M (SI Appendix, Table S1),the m-value for urea is expected to be ∼1.2 (20) and the Cm ofDi-III_14 is predicted to be ∼4.6 M urea (Fig. 1C). Its resistanceto unfolding up to 9.25 M urea implies a stability of greater than14 kcal/mol for a two-state process. To test the possible role of thesalt in modulating the resistance to urea denaturation, we added2.5 M NaCl to the solvent. As shown in Fig. 1C, Di-III_14 beginsto unfold at ∼7 M urea in the presence of 2.5 M salt, well abovethe expected Cm if urea and GdnHCl were to yield comparablemeasures of the stability. Screening of electrostatic interactionsalone does not account for the discrepancy between the urea andGdnHCl denaturation of Di-III_14. The very high apparentstability when measured by urea denaturation is consistent withits resistance to thermal unfolding, with a midpoint of equilibriumthermal denaturation >>95 °C (8).

Structural Correlates of Protection Against HDX in Di-III_14. To gainfurther insight into the contradictory perspectives on the foldingfree-energy surface of Di-III_14, we mapped the 29 stronglyprotected NHs onto the structure. We found that these NHscorrespond to a 15-residue hydrophobic core surrounded by 14solvent-exposed polar and charged side chains. Interestingly, 12of the latter 14 side chains are components of two electrostaticnetworks (Fig. 3). One spans the surface of α2 and links it to α1,and the other contains a quartet of salt bridges that link the twointernal β-strands, β2 and β4. The most slowly exchanging V31(β2) and I70 (β4) NHs are not only flanked by two of these saltbridges, but are also hydrogen-bonded to each other throughtheir amide NHs and carbonyl oxygens. The twofold slower ex-change for V31 vs. I70, however, shows that their shared H-bonds do not break in a concerted fashion (SI Appendix, Fig.S3 and Tables S2 and S3). The observed exchange-rate constantsare inversely correlated to the packing density (Fig. 4A) (21), asmight be expected for an EX1 mechanism whereby the molec-ular rearrangements required for exchange are limited by highbarriers. Comparisons with the average packing densities ofnatural mesophilic, thermophilic, and hyperthermophilic IF3-like proteins (Fig. 4B) find Di-III_14 to be as well packed asthe latter group of exceedingly stable proteins.Exchange-rate constants were uniformly accelerated by in-

creasing the concentration of NaCl by 0.25 M and 0.5 M (SIAppendix, Fig. S3). By contrast, nonmonotonic protection for NHsin α1, α2, β2, and β4, increasing and then decreasing, was observedwhen increasing the GdnHCl concentration from 0.5 to 1.0 M.The monotonic response to NaCl likely reflects the screeningof electrostatic interactions on the surface of Di-III_14. Thenonmonotonic response to GdnHCl suggests that the guanidinium

cation selectively binds in the vicinity of the electrostatic networksin the native conformation, slowing exchange, before favoringdenaturation and accelerating exchange. Crystallographic molec-ular dynamics simulations and solution-phase studies have previouslyrevealed the binding of GdnHCl to the surface of native proteins,before its preferential binding to the unfolded state (22–24).

Mutational Analysis of Electrostatic and Hydrophobic Networks inDi-III_14.Electrostatic networks. To test the role of electrostatic networks indefining the folding free-energy surface of Di-III_14, we targetedthe E32 and R69 salt bridge, linking β2 and β4, and R46 in α2,the central cationic charge surrounded by a ring of anionic sidechains in α2 (Fig. 3), for mutagenesis. In all three cases, theneutralization of the charge by mutation to alanine weakened theresistance to urea unfolding, with unfolding beginning at ap-proximately 5 M urea that was not complete by 9 M urea (SIAppendix, Fig. S5B). By contrast, the individual reversal of thecharges at all three positions had a dramatic effect on the sta-bility of the variants. The E32R, R46E, and R69E variants arereadily unfolded in urea with modest stabilities comparable tothose from the GdnHCl melts, 3.32–5.48 kcal/mol (Fig. 1D and SIAppendix, Fig. S5 A and B and Table S4). E32R and R46E retainnative-like secondary structure, but R69E loses approximately25% of the CD signal at 222 nm (SI Appendix, Fig. S6). Notably,all three charge-reversal variants retain a detectable fraction oftheir ellipticity at 222 nm in the urea-denatured state relative tothe GdnHCl-denatured state (Fig. 1D and SI Appendix, Fig. S5 A

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Fig. 2. (A) Exchange-rate constants measured at pH 6.5 (○) and pH 7.4 (●)plotted against the rate constants at pH 6.0 at 25 °C. The solid line indicatesthe response expected for an EX1 mechanism, and the dotted and dashedlines indicate the responses expected for an EX2 mechanism at pH 6.5 and7.4, respectively. (B) Exchange-rate constants at pH 7.4 of V31A (●) and E32R(○) are plotted against the rate constants at pH 6.5 at 25 °C. The solid lineindicates the response expected for an EX1 mechanism, and the dashed linesindicate the responses expected for an EX2 mechanism at pH 7.4 for the Di-III_14 variants.

Fig. 3. Structural correlates of exchange-rate constants at pH 7.4 and 25 °C. (A)Measurable exchange-rate constants are superimposed on a ribbon diagram ofthe NMR structure of Di-III_14 (Protein Data Bank ID code 2LN3). V31 and I70,which have observable exchange over a 3-d collection window, are shown inblack. The color scale from orange to blue follows the quintiles of the observ-able range of the rate constants as indicated. Weakly protected residues (2.5 ×10−4·s−1 ≤ kex < 8.3 × 10−4·s−1) are shown in red. (B) Networks of electrostaticinteractions spanning the surface of helix α2 and linked to helix α1 (Left) andlinking β2 and β4 (Right). The orange dashed lines indicate distances of less than8 Å between the carboxyl oxygens on aspartic acid or glutamic acid side chainsand nitrogens on arginine or lysine side chains. The images were created withthe PyMOL Molecular Graphics System (version 1.2r3pre; Schrödinger).

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and B). Thus, the estimates of stability from urea melts are a lowerbound on the difference in free energies between the native andfully unfolded states of this set of proteins.To further probe the E32/R69 electrostatic interaction, we

constructed the R69K variant to test the sensitivity of the energysurface to a change in sequence while conserving the charge. Theurea melt for R69K began at ∼3 M urea and was only partiallycomplete at 9 M urea (SI Appendix, Fig. S7). Lysine, a primaryamine, is not capable of maintaining the remarkable resistance ofDi-III_14 to urea denaturation observed for the WT protein.Evidently, the structure of the guanidinium moiety, not simply apositive charge, is crucial to the electrostatic network.The dramatic decrease in resistance to urea denaturation and

retention of structure for the E32R mutation led to an HDXNMR study to determine if the mutation altered the exchangeproperties. We found that the E32R variant retained the samepH-independent EX1 exchange kinetics as the WT protein (Fig.2B), albeit in a faster time range. Therefore, the surface roughnessof Di-III_14 in water can persist in the absence of its unusualresistance to urea denaturation.Hydrophobic network. The uniquely strong protection for V31 andI70, deeply buried in the hydrophobic network, motivated a mu-tational analysis of their roles in defining the folding free-energysurface of Di-III_14. The replacement of V31 or I70 with alaninehad a striking effect on the chemical denaturation profiles (Fig.1D and SI Appendix, Fig. S5A). The V31A and I70A variants nowunfold in urea and yield stabilities very similar to those for theirrespective GdnHCl titrations (Fig. 1D and SI Appendix, Fig. S5Aand Table S4). Remarkably, the HDX experiment on V31Arevealed a much-accelerated exchange process governed by anEX2 mechanism (Fig. 2B). It is astonishing that the deletion ofonly two methyl groups when valine is replaced by alanine issufficient to eliminate the surface roughness in Di-III_14.

DiscussionThe de novo design of proteins has been remarkably successful,with a variety now available for novel applications (6, 25–27).Failed sequences indicate the need for further refinement (28),but targeting the final structure, presumably the global minimumin free energy, has been a winning strategy. However, as wefound in the present study of a small βα protein, the underlyingfree-energy surface may be very complex. The determination ofthe molecular basis for these complexities proved to be in-structive for the next generation of design strategies.

Molecular Basis for the Complex Folding Free-Energy Surface of Di-III_14. The folding free-energy surface of Di-III_14 is remarkablymore complex than in almost all other proteins studied thus far.The surface as determined by a GdnHCl melt is very simple, withonly the native and unfolded states being highly populated andseparated by a single barrier. The complete resistance to ureadenaturation implies a stability far greater than that observed inGdnHCl, reflecting contributions from the extensive electrostaticand hydrophobic networks of side chains in this small βα protein.The free-energy surface in water is exceedingly rough, with mul-tiple high-energy states that slowly interconvert to enable exchangeof main-chain NHs with solvent (Fig. 5).We offer conjecture that the dramatically different responses

to the chemical denaturants reflect several properties of GdnHCl.First, the ionic nature of GdnHCl screens surface electrostaticinteractions that contribute to stability (Fig. 1C). Second, theguanidinium moiety binds selectively to the surface of Di-III_14 inthe vicinity of salt bridges whose cationic component is arginine(SI Appendix, Fig. S3). Direct competition between the denaturantand R30 and R32 would further weaken the electrostatic network.Third, GdnHCl is a more potent denaturant than urea, bindingmore tightly to exposed main-chain amide linkage in the unfoldedstate (29) and increasing the solubility of nonpolar side chains inwater (30). Point mutations in the electrostatic and hydrophobicnetworks can enable two-state unfolding in urea; however, re-sidual structure persists in the urea-denatured state. We presumethat this residual structure, along with the hydrophobic network,also stabilizes the high-energy states that enable HDX in water.GdnHCl unfolds these states and results in a simple two-statefolding reaction when extrapolated to water.Electrostatic networks. A significant fraction of the charged sidechains in Di-III_14 are involved in a pair of electrostatic networks,one on the solvent-exposed faces of α2 and α1 and the otherlinking β2 and β4 (Fig. 3). Charge reversal at E32 (β2) and R69(β4) eliminates the resistance to urea denaturation and revealscomparable stabilities to GdnHCl melts. Looking more closely atthe β2 and β4 strands, we find a striking segregation of charge(Fig. 3). β2 contains four anionic side chains in alternating posi-tions, D28, E30, E32, and D34, and β4 contains a correspondingnumber of cationic side chains, K73, R71, R69, and K67, thatform interstrand salt bridges. R69 bridges E30 and E32 in a three-way salt bridge. Placing a cationic side chain, R32, adjacent to E30

Fig. 5. The free-energy landscape of Di-III 14 at pH 7.4 and 25 °C in water,illustrating the barrier heights and energies of a few representativeexchange-competent states relative to the native state, N. The exchange-competent states are labeled by the residue whose amide hydrogen atomexchanges with the solvent. The data do not distinguish between stochasticand sequential mechanisms for exchange. The procedures used to calculatethe barrier heights and free energies of the exchange-competent states aredescribed in the SI Appendix. The relative free energy of the unfolded state,U, and the transition state to the native basin in water are not known andare represented as a dashed line.

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Fig. 4. (A) Correlation between the normalized packing densities of eachamino acid in WT Di-III_14 calculated with Voronoia software (21) and ob-served exchange-rate constants of their amide hydrogens at pH 7.4 and 25 °C.(B) Comparative global analysis of the normalized packing densities of natu-rally occurring IF3-like fold proteins from different mesophilic (37 °C), ther-mophilic (75 °C), and hyperthermophilic (95 °C) organisms with Di-III_14. Thewidth of the vertical line indicates the statistical dispersion, and the middlebar represents the median of the distribution. The small cross symbols rep-resent the outliers. The vertical brackets indicate the maximum and minimumvalues of packing densities. The small square boxes correspond to the repre-sentative averaged value of the packing density for the mesophilic (0.715),thermophilic (0.764), and hyperthermophilic (0.83) variants, respectively.

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and D34 in β2 and opposite R69 in β4 introduces intrastrand at-tractive interactions in β2 and repulsive interstrand interactionsbetween β2 and β4, effectively uncoupling the network that stabi-lizes the native conformation. The similar effects of replacing R69in β4 and R46 in α2 with an anionic glutamic acid reinforces theconclusion that both networks are crucial to the resistance to ureadenaturation. Several groups (31–33) have explored the relation-ship between electrostatic interactions on the surface of proteinsand their stabilities. Makhatadze and coworkers (31) have foundthat the optimization of electrostatic contributions to stability re-flects less the pair-wise short-range interactions and more thecombined effect of multiple long-range interactions defined by theglobal distribution of charges. The pair of electrostatic networks inDi-III_14 illustrates another source of collective interactions thatserve to enhance its stability. Although not an explicit feature ofthe de novo design, the effect on the stability to thermal or ureadenaturation is dramatic for such a small protein.Hydrophobic network. The strong protection against HDX in waterfor Di-III_14 also draws attention to a 15-residue network ofnonpolar side chains, FILV (phenylalanine-isoleucine-leucine-valine), sandwiched between the surface α-helices and the un-derlying β-sheet. Especially noteworthy are V31 and I70 that ex-change over a period of days. The replacement of the branchedaliphatic side chains in V31 or I70 with alanine eliminates theresistance to urea denaturation, demonstrating that the hydro-phobic network also plays a crucial role in the differential effectsof the denaturants. In contrast to the disruption of the electro-static network by charge reversal, the replacement of the isopropylside chain of V31 with the methyl group of alanine eliminates therough energy surface of Di-III_14. As the last NH to exchange,V31 must stabilize all of the preceding high-energy states on thefolding free-energy surface of Di-III_14 and support the barriersbetween these states that give rise to EX1 exchange kinetics.There are two scenarios for the EX1 HDX kinetics. One

scenario imagines a progressive unfolding of the protein, dic-tated by the packing density. The most deeply buried V31 is thelast to be exposed to solvent as its hydrophobic environmentmelts away. The second scenario imagines a stochastic exposureof the main-chain NHs, with the hydrophobic core largely intactbut dynamic, as each NH is uniquely exposed to solvent beforethe core reforms around it. The first scenario is favored by theobservation of residual secondary structure in urea-denaturedE32R (Fig. 1D and SI Appendix, Fig. S5A). The persistence ofresidual structure in 9.25 M urea for E32R makes it very likelythat residual structure also exists in the exchange-competentstate for V31 in the parent Di-III_14 sequence in water. Wepresume that this residual structure is the platform that stabilizesthe hydrophobic core and, in combination with the core, resultsin a rough energy surface. In either scenario, the inverse correlationof exchange-rate constants with packing density in the native con-formation means that exchange must take place from higher-energystates in the native basin. These exchange-competent states in thenative basin are separated by substantial barriers as a result ofthe large hydrophobic and electrostatic networks that charac-terize the structure of Di-III_14.In addition, the protection from hydrogen exchange observed at

0.5 M GdnHCl (SI Appendix, Fig. S3) is likely a result of the in-teraction of the guanidinium cation with the electrostatic net-works present in the native conformation. This observationfurther supports our hypothesis that the rugged folding freeenergy of Di-III_14, characterized by multiple exchange-competent states, exists in the native basin. Acceleration of theexchange process is observed at 1.0 M GdnHCl as the denatur-ation potential of the guanidinium cation begins to modulate thefree-energy surface.

Charge Segregation and the Folding Free-Energy Surface of Di-III_14.Das and Pappu (34) have simulated the behavior of polypeptidescontaining various compositions and distributions of oppositelycharged side chains, lysine and glutamic acid, and constructed aphase diagram for polyampholytes. They found that the total

fraction of charged residues, the net charge per residue (NCPR),and a normalized charge segregation parameter, κ, were suffi-cient to predict chain compaction. Di-III_14, with 35% chargedresidues, an NCPR of −0.06, and a κ value of 0.25, is expected toform a compact unfolded state in water (34, 35). The predictedcompactness derives in part from the preferred proximity ofoppositely charged segments in the unfolded state. Not consid-ered in their analysis was the potential effect of charge segre-gation on the native state of a resident protein. The dramaticeffect on urea denaturation by neutralizing or reversing thecharge of either partner in the E32/R69 salt bridge in Di-III_14points to a potential role for charge segregation in creatingelectrostatic networks that make significant contributions to thefree energy of the native state.

Implications from Evolution for Protein Design. The complex energysurface observed for Di-III_14 in water has implications for thedesign of proteins with folding landscapes more similar to thoseof naturally occurring proteins.Electrostatics. The unusually high but balanced composition ofcharged side chains and their segregation into positive andnegative segments of the sequence in Di-III_14 differentiate thisdesigned protein from almost the entire proteome of the ther-mophile Sulfolobus solfataricus (Fig. 6). Charge neutralization orreversal of individual residues that participate in the pair ofelectrostatic networks overcomes the complete resistance to ureadenaturation, resulting in an estimate of stability comparable tothat for GdnHCl denaturation, as seen for numerous other nat-urally occurring proteins. Decreasing the composition of chargedside chains and avoiding charge segregation to mimic thoseproperties found in proteomes whose sequences were filtered byevolution should be an important design feature.Hydrophobics. The sequence of Di-III_14 has a distinct basis towardlarger aliphatic side chains, 10 leucines and 6 isoleucines vs. 1valine and 1 alanine; larger polar/charged side chains, 10 glutamicacids and 6 glutamines vs. 6 aspartic acids and 2 asparagines; andaway from aromatic side chains, 3 phenylalanines, 1 tryptophan,0 tyrosines, and 0 histidines. The bias toward larger side chains invery stable thermophilic proteins has been observed long ago (36);however, the present study argues that there are limits to the

Fig. 6. Distribution of the normalized charge segregation parameter, κ, vs.the fraction of charged residues for the S. solfataricus proteome from Uni-Prot, calculated with the CIDER algorithm (34). Scatter-plot color coding isscaled from high disorder (red) to low disorder (blue). The bold red circlecorresponds to the value for Di_III-14. Representations of the integration ofthe scatter plots along the x-axis (Top) and y-axis (Right) are also shown.

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benefits of an increased packing density. The β- and γ-branchedside chains in the isoleucine–leucine–valine (ILV) subset can alsocreate an interlocked network that could impede interconver-sions between high-energy states and contribute to a rough energysurface. The introduction of even a single alanine in place of avaline, V31A, unlocks the hydrophobic network and allows therapid interchange between high-energy states in the native basin.Perhaps the lesson from evolution is that the inclusion of a morediverse set of nonpolar side chains in design, e.g., alanine, cysteine,and methionine, would enable the retention of the high packingdensity seen in thermophilic proteins (Fig. 4B), but reduce theprobability of an interlocked network of side chains that create therough energy surface seen for Di-III_14.The rough energy surface in this small designed protein, cre-

ated by a gradient in the packing density and enhanced by a pairof electrostatic networks, is a consequence of a design algorithmthat logically favors stability (i.e., packing density) and solubility(i.e., charged side chains on the surface). The lessons fromevolution—to increase the compositional complexity, reduce thecomposition of charged side chains, and avoid charge segregation—can readily be incorporated into future design projects.

Materials and MethodsProtein Expression and Purification. Di-III_14 was engineered with an N-terminal His6 tag and an intervening TEV Protease site (GenScript), ligatedinto the expression plasmid pGS-21a, and transfected into the Escherichia colistrain BL21 CodonPlus (DE3)RIL for expression. The purification processes areprovided in the SI Appendix.

CD and Fluorescence Analysis. Far-UV CD experiments were carried out in100 mM NaCl, 5.6 mM Na2HPO4, and 1.1 mM KH2PO4 buffer at different pHsat 25 °C on a JASCO model J810 CD spectrophotometer. Intrinsic tryptophanfluorescence was monitored on a Horiba Flourolog3 by using a 0.5-cm path

length, a slit width of 5 nm, and excitation at 295 nm. The fluorescence andCD data were globally fit to a two-state N ⇌ U model as a function of de-naturant by using our in-house Savuka and commercial Origin softwarepackage. More details about the fitting are provided in the SI Appendix.

Kinetic Studies. The time dependence of the Trp fluorescence intensity duringunfolding and refolding reactions was monitored in the millisecond timeregime on an Applied Photophysics SF system, model SX.18MV, with a deadtime of 2 ms. Di-III_14 was injected from stock concentrations of 40 μM andmixed with a 1:10 dilution to 4 μM with 100 mM NaCl, 5.6 mM Na2HPO4,1.1 mM KH2PO4 at varying pHs to the desired final experimental GdnHClconcentration.

Kinetic folding reactions in the microsecond time regime were measuredon a home-built CF time-correlated single photon counting instrument withmixing time of 60 μs. Further details are provided in the SI Appendix.

HDX NMR. Uniform labeling of Di-III_14 with 15N was accomplished by growingcells in isotopically enriched M9medium, 1 g 15NH4Cl per liter. The sample waslyophilized after purification. The experimental buffer used in the HDX NMRexperiments contained 100 mM NaCl, 5.6 mM Na2HPO4, and 1.1 mM KH2PO4

at pH 7.4, pH 6.5, or pH 6.0. All pH values reported are pD values obtained bycorrecting the pH meter readings: pD = pH (meter reading) + 0.41. Hydrogenexchange rates were measured in two ways: (i) lyophilized samples of 15NDi-III_14 were dissolved in a 100% D2O experimental buffer described earlier or(ii) samples of 15N Di-III_14 dissolved in 100% H2O experimental buffer werediluted in 100% D2O buffer to a final ratio of 90% D2O/10% H2O. Furtherdetails are provided in the SI Appendix.

ACKNOWLEDGMENTS. We thank all members of the laboratories of F.M.and C.R.M. for helpful discussions. This work was supported by NationalScience Foundation Grant MCB 1121942 (to C.R.M.), National Institutes ofHealth Grant GM117008 (to F.M.), and the Howard Hughes MedicalInstitute (D.B.).

1. Huang P-S, et al. (2016) De novo design of a four-fold symmetric TIM-barrel protein

with atomic-level accuracy. Nat Chem Biol 12:29–34.2. Keefe AD, Szostak JW (2001) Functional proteins from a random-sequence library.

Nature 410:715–718.3. Sillitoe I, et al. (2013) New functional families (FunFams) in CATH to improve the

mapping of conserved functional sites to 3D structures. Nucleic Acids Res 41:

D490–D498.4. Regan L, DeGrado WF (1988) Characterization of a helical protein designed from first

principles. Science 241:976–978.5. Harbury PB, Plecs JJ, Tidor B, Alber T, Kim PS (1998) High-resolution protein design

with backbone freedom. Science 282:1462–1467.6. Kuhlman B, et al. (2003) Design of a novel globular protein fold with atomic-level

accuracy. Science 302:1364–1368.7. Watters AL, et al. (2007) The highly cooperative folding of small naturally occurring

proteins is likely the result of natural selection. Cell 128:613–624.8. Koga N, et al. (2012) Principles for designing ideal protein structures. Nature 491:

222–227.9. Rocklin GJ, et al. (2017) Global analysis of protein folding using massively parallel

design, synthesis, and testing. Science 357:168–175.10. Bale JB, et al. (2016) Accurate design of megadalton-scale two-component icosahe-

dral protein complexes. Science 353:389–394.11. Boyken SE, et al. (2016) De novo design of protein homo-oligomers with modular

hydrogen-bond network–mediated specificity. Science 352:680–687.12. Lu P, et al. (2018) Accurate computational design of multipass transmembrane pro-

teins. Science 359:1042–1046.13. Butterfield GL, et al. (2017) Evolution of a designed protein assembly encapsulating

its own RNA genome. Nature 552:415–420.14. Plaxco KW, Simons KT, Ruczinski I, Baker D (2000) Topology, stability, sequence, and

length: Defining the determinants of two-state protein folding kinetics. Biochemistry

39:11177–11183.15. Matthews CR (1987) Effect of point mutations on the folding of globular proteins.

Methods Enzymol 154:498–511.16. Krishna MMG, Hoang L, Lin Y, Englander SW (2004) Hydrogen exchange methods to

study protein folding. Methods 34:51–64.17. Maity H, Lim WK, Rumbley JN, Englander SW (2003) Protein hydrogen exchange

mechanism: Local fluctuations. Protein Sci 12:153–160.18. Zhang YZ (1995) Protein and peptide structure and interactions studied by hydrogen

exchange and NMR. PhD thesis (Univ Pennsylvania, Philadelphia). Available at https://

books.google.com/books?id=p-KcNwAACAAJ.19. Pace CN (1990) Measuring and increasing protein stability. Trends Biotechnol 8:93–98.

20. Myers JK, Pace CN, Scholtz JM (1995) Denaturant m values and heat capacity changes:Relation to changes in accessible surface areas of protein unfolding. Protein Sci 4:2138–2148.

21. Rother K, Hildebrand PW, Goede A, Gruening B, Preissner R (2009) Voronoia: Ana-lyzing packing in protein structures. Nucleic Acids Res 37:D393–D395.

22. Gangadhara BN, Laine JM, Kathuria SV, Massi F, Matthews CR (2013) Clusters ofbranched aliphatic side chains serve as cores of stability in the native state of the HisFTIM barrel protein. J Mol Biol 425:1065–1081.

23. Gu Z, Zitzewitz JA, Matthews CR (2007) Mapping the structure of folding cores in TIMbarrel proteins by hydrogen exchange mass spectrometry: The roles of motif andsequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus.J Mol Biol 368:582–594.

24. Jha SK, Marqusee S (2014) Kinetic evidence for a two-stage mechanism of proteindenaturation by guanidinium chloride. Proc Natl Acad Sci USA 111:4856–4861.

25. Chevalier A, et al. (2017) Massively parallel de novo protein design for targetedtherapeutics. Nature 550:74–79.

26. Hosseinzadeh P, et al. (2017) Comprehensive computational design of ordered pep-tide macrocycles. Science 358:1461–1466.

27. Hsia Y, et al. (2016) Design of a hyperstable 60-subunit protein dodecahedron. [cor-rected]. Nature 535:136–139, and erratum (2016) 540:150.

28. Kuhlman B, Baker D (2000) Native protein sequences are close to optimal for theirstructures. Proc Natl Acad Sci USA 97:10383–10388.

29. Tanford C (1970) Protein Denaturation: Part C. Theoretical Models for the Mechanismof Denaturation. Advances in Protein Chemistry, eds Anfinsen CB, Edsall JT, Richards FM(Academic, New York), Vol 24, pp 1–95.

30. Nozaki Y, Tanford C (1965) The solubility of amino acids and related compounds inaqueous thylene glycol solutions. J Biol Chem 240:3568–3575.

31. Tzul FO, Schweiker KL, Makhatadze GI (2015) Modulation of folding energy land-scape by charge–Charge interactions: Linking experiments with computationalmodeling. Proc Natl Acad Sci USA 112:E259–E266.

32. Grimsley GR, et al. (1999) Increasing protein stability by altering long-range cou-lombic interactions. Protein Sci 8:1843–1849.

33. Spector S, et al. (2000) Rational modification of protein stability by the mutation ofcharged surface residues. Biochemistry 39:872–879.

34. Das RK, Pappu RV (2013) Conformations of intrinsically disordered proteins areinfluenced by linear sequence distributions of oppositely charged residues. Proc NatlAcad Sci USA 110:13392–13397.

35. Huihui J, Firman T, Ghosh K (2018) Modulating charge patterning and ionic strengthas a strategy to induce conformational changes in intrinsically disordered proteins.J Chem Phys 149:085101.

36. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: Sources, uses, and molecularmechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43.

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