The Energetics of Molecular Adaptation inTranscriptional RegulationGriffin Churea, Manuel Razo-Mejiaa, Nathan M. Belliveaua,b, Tal Einavc, Zofii A. Kaczmareka, Stephanie L. Barnesa,d, MitchellLewise, and Rob Phillipsa, f, 1

aDepartment of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA; bPresent address: Howard Hughes Medical Institute andDepartment of Biology, University of Washington, Seattle, WA, USA; cDepartment of Physics, California Institute of Technology, Pasadena, CA, USA; dPresent Address:Department of Science and Technology, Windward School, Los Angeles, CA, USA; eDepartment of Biochemistry and Molecular Biophysics, University of Pennsylvania Schoolof Medicine, Philadelphia, PA, USA; fDivision of Physics, Mathematics and Astronomy, California Insitute of Technology, Pasasdena, CA, USA

This manuscript was compiled on May 13, 2019

Mutation is a critical mechanism by which evolution explores thefunctional landscape of proteins. Despite our ability to experimen-tally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present aframework centered on measuring changes in the free energy of thesystem to link individual mutations in an allosteric transcriptional re-pressor to the parameters which govern its response. We find theenergetic effects of the mutations can be categorized into severalclasses which have characteristic curves as a function of the inducerconcentration. We experimentally test these diagnostic predictionsusing the well-characterized LacI repressor of Escherichia coli, prob-ing several mutations in the DNA binding and inducer binding do-mains. We find that the change in gene expression due to a pointmutation can be captured by modifying only a subset of the modelparameters that describe the respective domain of the wild-type pro-tein. These parameters appear to be insulated, with mutations inthe DNA binding domain altering only the DNA affinity and those inthe inducer binding domain altering only the allosteric parameters.Changing these subsets of parameters tunes the free energy of thesystem in a way that is concordant with theoretical expectations. Fi-nally, we show that the induction profiles and resulting free energiesassociated with pairwise double mutants can be predicted with quan-titative accuracy given knowledge of the single mutants, providingan avenue for identifying and quantifying epistatic interactions.

Transcriptional Regulation | Allostery | MWC Model | Statistical Mechanics |Epistasis | Biophysics | Evolution | Mutation

Thermodynamic treatments of transcriptional regulationhave been fruitful in their ability to generate quantitativepredictions of gene expression as a function of a minimal set ofphysically meaningful variables (1–13). These models quantita-tively describe numerous properties of input-output functions,such as the leakiness, saturation, dynamic range, steepnessof response, and the [EC50] – the concentration of inducer atwhich the response is half maximal. The mathematical formsof these phenotypic properties are couched in terms of a min-imal set of experimentally accessible variables, such as theinducer concentration, transcription factor copy number, andthe DNA sequence of the binding site (10). While the aminoacid sequence of the transcription factor is another controllablevariable, it is seldom implemented in quantitative terms con-sidering mutations with subtle changes in chemistry frequentlyresult in unpredictable physiological consequences. In thiswork, we examine how a series of mutations in either the DNAbinding or inducer binding domains of a transcriptional repres-sor influence the values of the biophysical parameters whichgovern its regulatory behavior.

We first present a theoretical framework for understandinghow mutations in the repressor affect different parameters andalter the free energy of the system. The multi-dimensional pa-rameter space of the aforementioned thermodynamic modelsis highly degenerate with multiple combinations of parametervalues yielding the same phenotypic response. This degeneracycan be subsumed into the free energy of the system, transform-ing the input-output function into a one-dimensional descrip-tion with the form of a Fermi function (14, 15). We find thatthe parameters capturing the allosteric nature of the repressor,the repressor copy number, and the DNA binding specificitycontribute independently to the free energy of the system withdifferent degrees of sensitivity. Furthermore, changes restrictedto one of these three groups of parameters result in characteris-tic changes in the free energy relative to the wild-type repressor,providing falsifiable predictions of how different classes of mu-tations should behave.

Next, we test these descriptions experimentally using thewell-characterized transcriptional repressor of the lac operonLacI in E. coli regulating expression of a fluorescent reporter.We introduce a series of point mutations in either the inducerbinding or DNA binding domain. We then measure the fullinduction profile of each mutant, determine the minimal setof parameters that are affected by the mutation, and predicthow each mutation tunes the free energy at different inducerconcentrations, repressor copy numbers, and DNA binding

Summary

We present a biophysical model of allosteric transcriptional reg-ulation that directly links the location of a mutation within a re-pressor to the biophysical parameters that describe its behavior.We explore the phenotypic space of a repressor with mutationsin either the inducer binding or DNA binding domains. Using theLacI repressor in E. coli, we make sharp, falsifiable predictionsand use this framework to generate a null hypothesis for howdouble mutants behave given knowledge of the single mutants.Linking mutations to the parameters which govern the systemallows for quantitative predictions of how the free energy ofthe system changes as a result, permitting coarse graining ofhigh-dimensional data into a single-parameter description ofthe mutational consequences.

The authors declare no conflict of interest.

1To whom correspondence should be addressed. E-mail: phillips@pboc.caltech.edu

strengths. We find in general that mutations in the DNA bind-ing domain only influence DNA binding strength, and thatmutations within the inducer binding domain affect only theparameters which dictate the allosteric response. The degreeto which these parameters are insulated is notable, as the verynature of allostery suggests that all parameters are intimatelyconnected, thus enabling binding events at one domain to be"sensed" by another.

With knowledge of how a collection of DNA binding andinducer binding single mutants behave, we predict the induc-tion profiles and the free energy changes of pairwise doublemutants with quantitative accuracy. We find that the energeticeffects of each individual mutation are additive, indicating thatepistatic interactions are absent between the mutations exam-ined here. Our model provides a means for identifying andquantifying the extent of epistatic interactions in a more com-plex set of mutations, and can shed light on how the proteinsequence and general regulatory architecture coevolve.

Results

This work considers the inducible simple repression regulatorymotif [depicted in Fig. 1(A)] from a thermodynamic perspectivewhich has been thoroughly dissected and tested experimentally(4, 6, 10). While we direct the reader to the SI text for a completederivation, the result of this extensive theory-experiment dia-logue is a succinct input-output function [schematized in Fig.1(B)] that computes the fold-change in gene expression relativeto an unregulated promoter. This function is of the form

fold-change =✓

1 +RA

NNSe�bD#RA

◆�1, [1]

where RA is the number of active repressors per cell, NNS is thenumber of non-specific binding sites for the repressor, D#RA isthe binding energy of the repressor to its specific binding siterelative to the non-specific background, and b is defined as 1kBTwhere kB is the Boltzmann constant and T is the temperature.While this theory requires knowledge of the number of activerepressors, we often only know the total number R which isthe sum total of active and inactive repressors. We can definea prefactor pact(c) which captures the allosteric nature of therepressor and encodes the probability a repressor is in the ac-tive (repressive) state rather than the inactive state for a giveninducer concentration c, namely,

pact(c) =

⇣1 + cKA

⌘n

⇣1 + cKA

⌘n+ e�bD#AI

⇣1 + cKI

⌘n . [2]

Here, KA and KI are the dissociation constants of the inducerto the active and inactive repressor, D#AI is the energetic differ-ence between the repressor active and inactive states, and n isthe number of allosteric binding sites per repressor molecule(n = 2 for LacI). With this in hand, we can define RA in Eq. (1)as RA = pact(c)R.

A key feature of Eq. (1) and Eq. (2) is that the diverse phe-nomenology of the gene expression induction profile can becollapsed onto a single master curve by rewriting the input-output function in terms of the free energy F [also called theBohr parameter (16)],

fold-change =⇣

1 + e�bF⌘�1

, [3]

where

F = �kBT log pact(c)� kBT log✓

RNNS

◆+ D#RA. [4]

Hence, if different combinations of parameters yield the samefree energy, they will give rise to the same fold-change in geneexpression, enabling us to collapse multiple regulatory scenar-ios onto a single curve. This can be seen in Fig. 1(C) whereeighteen unique inducer titration profiles of a LacI simple re-pression architecture collected and analyzed in Razo-Mejia et al.2018 (10) collapse onto a single master curve. The tight distribu-tion about this curve reveals that fold-change across a varietyof genetically distinct individuals can be adequately describedby a small number of parameters. Beyond predicting the induc-tion profiles of different strains, the method of data collapseinspired by Eq. (3) and Eq. (4) can be used as a tool to identifymechanistic changes in the regulatory architecture (14). Similardata collapse approaches have been used previously in sucha manner and have proved vital for distinguishing betweenchanges in parameter values and changes in the fundamentalbehavior of the system (14, 15).

Assuming that a given mutation does not result in a non-functional protein, it is reasonable to say that any or all of theparameters in Eq. (1) can be affected by the mutation, changingthe observed induction profile and therefore the free energy. Toexamine how the free energy of a mutant F(mut) differs fromthat of the wild-type F(wt), we define DF = F(mut) � F(wt),which has the form

DF = �kBT log

p(mut)act (c)

p(wt)act (c)

!� kBT log

R(mut)

R(wt)

!

+ (D#(mut)RA � D#(wt)RA ).

[5]

DF describes how a mutation translates a point across themaster curve shown in Fig. 1(C). As we will show in the comingparagraphs [illustrated in Fig. 2], this formulation coarse grainsthe myriad parameters shown in Eq. (1) and Eq. (2) into threedistinct quantities, each with different sensitivities to paramet-ric changes. By examining how a mutation changes the freeenergy changes as a function of the inducer concentration, onecan draw conclusions as to which parameters have been modi-fied based solely on the shape of the curve. To help the readerunderstand how various perturbations to the parameters tunethe free energy, we have hosted an interactive figure on thepaper website which makes exploration of parameter space asimpler task.

The first term in Eq. (5) is the log ratio of the probability of amutant repressor being active relative to the wild type at a giveninducer concentration c. This quantity defines how changesto any of the allosteric parameters – such as inducer bindingconstants KA and KI , or active/inactive state energetic differ-ence D#AI – alter the free energy F, which can be interpretedas the free energy difference between the repressor bound andunbound states of the promoter. Fig. 2 (A) illustrates howchanges to the inducer binding constants KA and KI alone alterthe induction profiles and resulting free energy as a function ofthe inducer concentration. In the limit where c = 0, the valuesof KA and KI do not factor into the calculation of pact(c) givenby Eq. (2), meaning that D#AI is the lone parameter setting theresidual activity of the repressor. Thus, if only KA and KI arealtered by a mutation, then DF should be 0 kBT when c = 0,

2

http://www.rpgroup.caltech.edu/mwc_mutants

inducer concentration

fold-change ingene expression

repressor copynumber

DNA bindingenergy

active repressorinducer dissociation

constant

inactive repressorinducer dissociation

constant

allosteric energy

differencefr

ee e

nerg

y

∆ε

A PREDICTIVE THEORY OF ALLOSTERIC INDUCTION PHENOTYPIC DATA COLLAPSE

RNA polymerase

activerepressor

inactiverepressor

inducer

THE SIMPLE REPRESSION ARCHITECTURE

(A) (B) (C)

free energy

+

free

ene

rgy

Fig. 1. A predictive framework for phenotypic and energetic dissection of the simple repression motif. (A) The inducible simple repression architecture. When in the active state,the repressor (gray) binds the cognate operator sequence of the DNA (red box) with high specificity, preventing transcription by occluding binding of the RNA polymerase to thepromoter (blue rectangle). Upon addition of an inducer molecule, the inactive state becomes energetically preferable and the repressor no longer binds the operator sequencewith appreciable specificity. Once unbound from the operator, binding of the RNA polymerase (blue) is no longer blocked and transcription can occur. (B) The simple repressioninput-output function for an allosteric repressor with two inducer binding sites. The key parameters are identified in speech bubbles. (C) Fold-change in gene expressioncollapses as a function of the free energy. The input-output function in (B) can be re-written as a Fermi function with an energetic parameter F which is the energetic differencebetween the repressor bound and unbound states of the promoter. Top panel shows induction profiles reported in Razo-Mejia et al. 2018 (10) of eighteen different strains overtwelve concentrations of the gratuitous inducer Isopropyl b-D-1-thiogalactopyranoside (IPTG). Upon calculation of the free energy, the data collapse onto a single master curve(bottom panel) defined by F.

illustrated by the overlapping red, purple, and grey curves inthe right-hand plot of Fig. 2(A). However, if D#AI is influencedby the mutation (either alone or in conjunction with KA andKI), the leakiness will change, resulting in a non-zero DF whenc = 0. This is illustrated in Fig. 2 (B) where D#AI is the onlyparameter affected by the mutation.

It is important to note that for a mutation which perturbsonly the inducer binding constants, the dependence of DF onthe inducer concentration can be non-monotonic. While theprecise values of KA and KI control the sensitivity of the repres-sor to inducer concentration, it is the ratio KA/KI that defineswhether this non-monotonic behavior is observed. This canbe seen more clearly when we consider the limit of saturatinginducer concentration,

limc!•

log

p(mut)actp(wt)act

!⇡ log

2

664

1 + e�bD#(wt)AI

✓K(wt)AK(wt)I

◆n

1 + e�bD#(wt)AI

✓K(mut)AK(mut)I

◆n

3

775 , [6]

which illustrates that DF returns to zero at saturating inducerconcentration when KA/KI is the same for both the mutantand wild-type repressors, so long as D#AI is unperturbed. Non-monotonicity can only be achieved by changing KA and KIand therefore serves as a diagnostic for classifying mutationaleffects reliant solely on measuring the change in free energy.

The second term in Eq. (5) captures how changes in the re-pressor copy number contributes to changes in free energy. Itis important to note that this contribution to the free energychange depends on the total number of repressors in the cell,

not just those in the active state. This emphasizes that changesin the expression of the repressor are energetically divorcedfrom changes to the allosteric nature of the repressor. As a con-sequence, the change in free energy is constant for all inducerconcentrations, as is schematized in Fig. 2(C). Because magni-tude of the change in free energy scales logarithmically withchanging repressor copy number, a mutation which increasesexpression from 1 to 10 repressors per cell is more impactfulfrom an energetic standpoint (kBT log(10) ⇡ 2.3 kBT) than anincrease from 90 to 100 (kBT log(100/90) ⇡ 0.1 kBT). Appre-ciable changes in the free energy only arise when variations inthe repressor copy number are larger than or comparable to anorder of magnitude. Changes of this magnitude are certainlypossible from a single point mutation, as it has been shownthat even synonymous substitutions can drastically changetranslation efficiency (17).

The third and final term in Eq. (5) is the difference in theDNA binding energy between the mutant and wild-type re-pressors. All else being equal, if the mutated state binds moretightly to the DNA than the wild type (D#(wt)RA > D#

(mut)RA ), the

net change in the free energy is negative, indicating that the re-pressor bound states become more energetically favorable dueto the mutation. Much like in the case of changing repressorcopy number, this quantity is independent of inducer concen-tration and is therefore also constant [Fig. 2(D)]. However, themagnitude of the change in free energy is linear with DNAbinding affinity while it is logarithmic with respect to changesin the repressor copy number. Thus, to change the free energyby 1 kBT, the repressor copy number must change by a factor of

3

affected parameter change in free energy

< <

<

< <

<

< <

inducer concentration

∆ F

inducer concentration

∆ F

inducer concentration

∆ F

inducer concentration

fold

-cha

nge

free energy

∆ F

fold

-cha

nge

free energy

fold

-cha

nge

free energy

fold

-cha

nge

free energy

0

∆F < 0

12 3

1

3

Fig. 2. Parametric changes due to mutations alter the free energy. The first column schematizes the changed parameters and the second column reflects which quantity inEq. (5) is affected. The third column shows representative induction profiles from mutants which have smaller (red) and larger (purple) values for the parameters than thewild-type (grey). The fourth and fifth columns illustrate how the free energy is changed as a result. Purple and red arrows indicate the direction in which the points are translatedabout the master curve. Three concentrations (points labeled 1, 2, and 3) are shown to illustrate how each point is moved in free energy space.

⇡ 2.3 whereas the DNA binding energy must change by 1 kBT.The unique behavior of each quantity in Eq. (5) and its sen-

sitivity with respect to the parameters makes DF useful as adiagnostic tool to classify mutations. Given a set of fold-changemeasurements, a simple rearrangement of Eq. (3) permits thedirect calculation of the free energy, assuming that the under-lying physics of the regulatory architecture has not changed.Thus, it becomes possible to experimentally test the generalassertions made in Fig. 2.

DNA Binding Domain Mutations. With this arsenal of analyticdiagnostics, we can begin to explore the mutational space ofthe repressor and map these mutations to the biophysical pa-rameters they control. As one of the most thoroughly studiedtranscription factors, LacI has been subjected to numerous crys-tallographic and mutational studies (18–21). One such workgenerated a set of point mutations in the LacI repressor andexamined the diversity of the phenotypic response to differentallosteric effectors (5). However, experimental variables suchas the repressor copy number or the number of specific bind-ing sites were not known, making precise calculation of DF aspresented here not tractable. Using this dataset as a guide, wechose a subset of the mutations and inserted them into our ex-perimental strains of E. coli where these parameters are knownand tightly controlled (4, 10).

We made three amino acid substitutions (Y20I, Q21A, andQ21M) that are critical for the DNA-repressor interaction. Thesemutations were introduced into the lacI sequence used in Garciaand Phillips 2011 (4) with four different ribosomal binding site

sequences that were shown (via quantitative Western blotting)to tune the wild-type repressor copy number across three ordersof magnitude. These mutant constructs were integrated intothe E. coli chromosome harboring a Yellow Fluorescent Protein(YFP) reporter. The YFP promoter included the native O2 LacIoperator sequence which the wild-type LacI repressor bindswith high specificity (D#RA = �13.9 kBT). The fold-change ingene expression for each mutant across twelve concentrationsof IPTG was measured via flow cytometry. As we mutatedonly a single amino acid with the minimum number of basepair changes to the codons from the wild-type sequence, wefind it unlikely that the repressor copy number was drasticallyaltered from those reported in (4) for the wild-type sequencepaired with the same ribosomal binding site sequences. Incharacterizing the effects of these DNA binding mutations, wetake the repressor copy number to be unchanged. Any errorintroduced by this mutation should be manifest as a larger thanpredicted systematic shift in the free energy change when therepressor copy number is varied.

A naïve hypothesis for the effect of a mutation in the DNAbinding domain is that only the DNA binding energy is altered.This hypothesis appears to contradict the core principle of al-lostery in that ligand binding in one domain influences bindingin another, suggesting that changing any parameter modifiesthem all. The characteristic curves summarized in Fig. 2 givea means to discriminate between these two hypotheses by ex-amining the change in the free energy. Using a single inductionprofile (white-faced points in Fig. 3), we estimated the DNAbinding energy using a Bayesian approach, the details of which

4

Fig. 3. Induction profiles and free energy modifications of DNAbinding domain mutations. Each column corresponds to thehighlighted mutant at the top of the figure. Each strain waspaired with the native O2 operator sequence. White-faced pointscorrespond to the strain for each mutant from which the DNAbinding energy was estimated. (A) Induction profiles of eachmutant at four different repressor copy numbers as a functionof the inducer concentration. Points correspond to the meanfold-change in gene expression of six to ten biological replicates.Error bars are the standard error of the mean. Shaded regionsdemarcate the 95% credible region of the induction profile gen-erated by the estimated DNA binding energy. (B) Data collapseof all points for each mutant shown in (A) using only the DNAbinding energy estimated from a single repressor copy number.Points correspond to the average fold-change in gene expres-sion of six to ten biological replicates. Error bars are standarderror of the mean. Where error bars are not visible, the relativeerror in measurement is smaller than the size of the marker.(C) The change in the free energy resulting from each mutationas a function of the inducer concentration. Points correspondto the median of the marginal posterior distribution for the freeenergy. Error bars represent the upper and lower bounds ofthe 95% credible region. Points in (A) at the detection limits ofthe flow cytometer (near fold-change values of 0 and 1) wereneglected for calculation of the DF. The IPTG concentration isshown on a symmetric log scale with linear scaling ranging from0 to 10�2 µM and log scaling elsewhere.

are discussed in the Materials and Methods as well as the SI text.The shaded red region for each mutant in Fig. 3 represents the95% credible region of this fit whereas all other shaded regionsare 95% credible regions of the predictions for other repressorcopy numbers. We find that redetermining only the DNA bind-ing energy accurately captures the majority of the inductionprofiles, indicating that other parameters are unaffected. Oneexception is for the lowest repressor copy numbers (R = 60and R = 124 per cell) of mutant Q21A at low concentrationsof IPTG. However, we note that this disagreement is compara-ble to that observed for the wild-type repressor binding to theweakest operator in Razo-Mejia et al. 2018 (10), illustrating thatour model is imperfect in characterizing weakly repressing ar-chitectures. Including other parameters in the fit (such as D#AI)does not significantly improve the accuracy of the predictions.Furthermore, the magnitude of this disagreement also dependson the choice of the fitting strain (see SI text).

Mutations Y20I and Q21A both weaken the affinity ofthe repressor to the DNA relative to the wild type strain(�9.9+0.1�0.1 kBT and �11.0

+0.1�0.1 kBT, respectively). Here we report

the median of the inferred posterior probability distributionwith the superscripts and subscripts corresponding to the upperand lower bounds of the 95% credible region. These binding en-ergies are comparable to that of the wild-type repressor affinityto the native LacI operator sequence O3, with a DNA bindingenergy of �9.7 kBT. The mutation Q21M increases the strengthof the DNA-repressor interaction relative to the wild-type re-pressor with a binding energy of �15.43+0.07�0.06 kBT, comparableto the affinity of the wild-type repressor to the native O1 opera-tor sequence (�15.3kBT). It is notable that a single amino acidsubstitution of the repressor is capable of changing the strengthof the DNA binding interaction well beyond that of many singlebase-pair mutations in the operator sequence (4, 22).

Using the new DNA binding energies, we can collapse allmeasurements of fold-change as a function of the free energy

as shown in Fig. 3(B). This allows us to test the diagnosticpower of the decomposition of the free energy described in Fig.2. To compute the DF for each mutation, we inferred the ob-served mean free energy of the mutant strain for each inducerconcentration and repressor copy number (see Materials andMethods as well as the SI text for a detailed explanation of theinference). We note that in the limit of extremely low or highfold-change, the inference of the free energy is either over- orunder-estimated, respectively, introducing a systematic error.Thus, points which are close to these limits are omitted in thecalculation of DF. We direct the reader to the SI text for a de-tailed discussion of this systematic error. With a measure ofF(mut) for each mutant at each repressor copy number, we com-pute the difference in free energy relative to the wild-type strainwith the same repressor copy number and operator sequence,restricting all variability in DF solely to changes in D#RA.

The change in free energy for each mutant is shown in Fig.3(C). It can be seen that the DF for each mutant is constantas a function of the inducer concentration and is concordantwith the prediction generated from fitting D#RA to a singlerepressor copy number [red lines Fig. 3(C)]. This is in line withthe predictions outlined in Fig. 2(C) and (D), indicating thatthe allosteric parameters are "insulated", meaning they are notaffected by the DNA binding domain mutations. As the DF forall repressor copy numbers collapses onto the prediction, wecan say that the expression of the repressor itself is the sameor comparable with that of the wild type. If the repressor copynumber were perturbed in addition to D#RA, one would expecta shift away from the prediction that scales logarithmically withthe change in repressor copy number. However, as the DF isapproximately the same for each repressor copy number, it canbe surmised that the mutation does not significantly change theexpression or folding efficiency of the repressor itself. Theseresults allow us to state that the DNA binding energy D#RA isthe only parameter modified by the DNA mutants examined.

5

Fig. 4. Induction profiles and free energy differences of inducer binding domain mutants. White faced points represent the strain to which the parameters were fit, namely theO2 operator sequence. Each column corresponds to the mutant highlighted at the top of the figure. All strains have R = 260 per cell. (A) The fold-change in gene expression asa function of the inducer concentration for three operator sequences of varying strength. Dashed lines correspond to the curve of best fit resulting from fitting KA and KI alone.Shaded curves correspond to the 95% credible region of the induction profile determined from fitting KA , KI , and D#AI . Points correspond to the mean measurement of six totwelve biological replicates. Error bars are the standard error of the mean. (B) Points in (A) collapsed as a function of the free energy calculated from redetermining KA , KI , andD#AI . (C) Change in free energy resulting from each mutation as a function of the inducer concentration. Points correspond to the median of the posterior distribution for thefree energy. Error bars represent the upper and lower bounds of the 95% credible region. Shaded curves are the predictions. IPTG concentration is shown on a symmetric logscaling axis with the linear region spanning from 0 to 10�2 µM and log scaling elsewhere.

Inducer Binding Domain Mutations. Much as in the case of theDNA binding mutants, we cannot safely assume a priori that agiven mutation in the inducer binding domain affects only theinducer binding constants KA and KI . While it is easy to asso-ciate the inducer binding constants with the inducer bindingdomain, the critical parameter in our allosteric model D#AI isharder to restrict to a single spatial region of the protein. AsKA, KI , and D#AI are all parameters dictating the allosteric re-sponse, we consider two hypotheses in which inducer bindingmutations alter either all three parameters or only KA and KI .

We made four point mutations within the inducer bindingdomain of LacI (F164T, Q294V, Q294R, and Q294K) that havebeen shown previously to alter binding to multiple allostericeffectors (5). In contrast to the DNA binding domain mutants,we paired the inducer binding domain mutations with the threenative LacI operator sequences (which have various affinitiesfor the repressor) and a single ribosomal binding site sequence.This ribosomal binding site sequence, as reported in (4), ex-presses the wild-type LacI repressor to an average copy numberof approximately 260 per cell. As the free energy differencesresulting from point mutations in the DNA binding domaincan be described solely by changes to D#RA, we continue underthe assumption that the inducer binding domain mutations donot significantly alter the repressor copy number.

The induction profiles for these four mutants are shown inFig. 4(A). Of the mutations chosen, Q294R and Q294K appearto have the most significant impact, with Q294R abolishing the

characteristic sigmoidal titration curve entirely. It is notablethat both Q294R and Q294K have elevated expression in theabsence of inducer compared to the other two mutants pairedwith the same operator sequence. Panel (A) in Fig. 2 illustratesthat if only KA and KI were being affected by the mutations,the fold-change should be identical for all mutants in the ab-sence of inducer. This discrepancy in the observed leakinessimmediately suggests that more than KA and KI are affectedfor Q294K and Q294R.

Using a single induction profile for each mutant (shownin Fig. 4 as white-faced circles), we inferred the parametercombinations for both hypotheses and drew predictions forthe induction profiles with other operator sequences. We findthat the simplest hypothesis (in which only KA and KI arealtered) does not permit accurate prediction of most inductionprofiles. These curves, shown as dotted lines in Fig. 4(A), failspectacularly in the case of Q294R and Q294K, and undershootthe observed profiles for F164T and Q294V, especially whenpaired with the weak operator sequence O3. The change inthe leakiness for Q294R and Q294K is particularly evident asthe expression at c = 0 should be identical to the wild-typerepressor under this hypothesis. Altering only KA and KI is notsufficient to accurately predict the induction profiles for F164Tand Q294V, but not to the same degree as Q294K and Q294R.The disagreement is most evident for the weakest operator O3[green lines in Fig. 4(A)], though we have discussed previouslythat the induction profiles for weak operators are difficult to

6

Table 1. Inferred values of KA, KI , and D#AI for inducer binding mu-tants

Mutant KA KI D#AI [kBT] Reference

WT 139+29�22 µM 0.53+0.04�0.04 µM 4.5 (10)

F164T 165+90�65 µM 3+6�3 µM 1

+5�2 This study

Q294V 650+450�250 µM 8+8�8 µM 3

+6�3 This study

Q294K > 1 mM 310+70�60 µM �3.11+0.07�0.07 This study

Q294R 9+20�9 µM 8+20�8 µM �2.35

+0.01�0.09 This study

accurately describe and can result in comparable disagreementfor the wild-type repressor (10, 22).

Including D#AI as a perturbed parameter in addition to KAand KI improves the predicted profiles for all four mutants. Byfitting these three parameters to a single strain, we are able toaccurately predict the induction profiles of other operators asseen by the shaded lines in Fig. 4(A). With these modified pa-rameters, all experimental measurements collapse as a functionof their free energy as prescribed by Eq. (3) [Fig. 4(B)]. All fourmutations significantly diminish the binding affinity of bothstates of the repressor to the inducer, as seen by the estimatedparameter values reported in Tab. 1. As evident in the dataalone, Q294R abrogates inducibility outright (KA ⇡ KI). ForQ294K, the active state of the repressor can no longer bind in-ducer whereas the inactive state binds with weak affinity. Theremaining two mutants, Q294V and F164T, both show dimin-ished binding affinity of the inducer to both the active andinactive states of the repressor relative to the wild-type.

Given the collection of fold-change measurements, we com-puted the DF relative to the wild-type strain with the sameoperator and repressor copy number. This leaves differencesin pact(c) as the sole contributor to the free energy difference,assuming our hypothesis that KA, KI , and D#AI are the onlyperturbed parameters is correct. The change in free energy canbe seen in Fig. 4(C). For all mutants, the free energy differenceinferred from the observed fold-change measurements fallswithin error of the predictions generated under the hypothesisthat KA, KI , and D#AI are all affected by the mutation [shadedcurves in Fig. 4(C)]. The profile of the free energy change ex-hibits some of the rich phenomenology illustrated in Fig. 2(A)and (B). Q294K, F164T, and Q294V exhibit a non-monotonicdependence on the inducer concentration, a feature that canonly appear when KA and KI are altered. The non-zero DF atc = 0 for Q294R and Q294K coupled with an inducer concentra-tion dependence is a telling sign that D#AI must be significantlymodified. This shift in DF is positive in all cases, indicatingthat D#AI must have decreased, and that the inactive state hasbecome more energetically favorable for these mutants thanfor the wild-type protein. Indeed the estimates for D#AI (Tab.1) reveal both mutations Q294R and Q294K make the inactivestate more favorable than the active state. Thus, for these twomutations, only ⇡ 10% of the repressors are active in the ab-sence of inducer, whereas the basal active fraction is ⇡ 99% forthe wild-type repressor (10).

Taken together, these parametric changes diminish the re-sponse of the regulatory architecture as a whole to changing in-

ducer concentrations. They furthermore reveal that the param-eters which govern the allosteric response are interdependentand no single parameter is insulated from the others. How-ever, as only the allosteric parameters are changed, one can saythat the allosteric parameters as a whole are insulated from theother components which define the regulatory response, suchas repressor copy number and DNA binding affinity.

Predicting Effects of Pairwise Double Mutations. Given fullknowledge of each individual mutation, we can draw predic-tions of the behavior of the pairwise double mutants with nofree parameters based on the simplest null hypothesis of noepistasis. The formalism of DF defined by Eq. (5) explicitlystates that the contribution to the free energy of the systemfrom the difference in DNA binding energy and the allostericparameters are strictly additive. Thus, deviations from thepredicted change in free energy would suggest epistatic inter-actions between the two mutations.

To test this additive model, we constructed nine doublemutant strains, each having a unique inducer binding (F164T,Q294V, Q294K) and DNA binding mutation (Y20I, Q21A,Q21M). To make predictions with an appropriate representa-tion of the uncertainty, we computed a large array of inductionprofiles given random draws from the posterior distributionfor the DNA binding energy (determined from the single DNAbinding mutants) as well as from the joint posterior for theallosteric parameters (determined from the single inducer bind-ing mutants). These predictions, shown in Fig. 5(A) and (B)as shaded blue curves, capture all experimental measurementsof the fold-change [Fig. 5(A)] and the inferred difference infree energy [Fig. 5(B)]. The latter indicates that there are noepistatic interactions between the mutations queried in thiswork, though if there were, systematic deviations from thesepredictions would shed light on how the epistasis is manifest.

The precise agreement between the predictions and mea-surements for Q294K paired with either Q21A or Q21M is strik-ing as Q294K drastically changed D#AI in addition to KA andKI . Our ability to predict the induction profile and free en-ergy change underscores the extent to which the DNA bindingenergy and the allosteric parameters are insulated from oneanother. Despite this insulation, the repressor still functionsas an allosteric molecule, emphasizing that the mutations wehave inserted do not alter the pathway of communication be-tween the two domains of the protein. As the double mutantY20I-Q294K exhibits fold-change of approximately 1 across allIPTG concentrations [Fig. 5(A)], these mutations in tandemmake repression so weak it is beyond the limits which are de-tectable by our experiments. As a consequence, we are unableto estimate DF nor experimentally verify the correspondingprediction [grey box in Fig. 5(B)]. However, as the predictedfold-change in gene expression is also approximately 1 for all c,we believe that the prediction shown for DF is likely accurate.One would be able to infer the DF to confirm these predictionsusing a more sensitive method for measuring the fold-change,such as single-cell microscopy or colorimetric assays.

Discussion. Allosteric regulation is often couched as “biologi-cal action at a distance". Despite extensive knowledge of pro-tein structure and function, it remains difficult to translate thecoordinates of the atomic constituents of a protein to the pre-cise parameter values which define the functional response,making each mutant its own intellectual adventure. Bioin-

7

Fig. 5. Induction and free energy profiles of DNA binding and inducer binding double mutants.(A) Fold-change in gene expression for each double mutant as a function ofIPTG. Points and errors correspond to the mean and standard error of six to ten biological replicates. Where not visible, error bars are smaller than the corresponding marker.Shaded regions correspond to the 95% credible region of the prediction given knowledge of the single mutants. These were generated by drawing 104 samples from the D#RAposterior distribution of the single DNA binding domain mutants and the joint probability distribution of KA , KI , and D#AI from the single inducer binding domain mutants. (B)The difference in free energy of each double mutant as a function of the reference free energy. Points and errors correspond to the median and bounds of the 95% credibleregion of the posterior distribution for the inferred DF. Shaded lines region are the predicted change in free energy, generated in the same manner as the shaded lines in (A). Allmeasurements were taken from a strain with 260 repressors per cell paired with a reporter with the native O2 LacI operator sequence. In all plots, the IPTG concentration isshown on a symmetric log axis with linear scaling between 0 and 10�2 µM and log scaling elsewhere.

formatic approaches to understanding the sequence-structurerelationship have permitted us to examine how the residues ofallosteric proteins evolve, revealing conserved regions whichhint to their function. Co-evolving residues reveal sectors ofconserved interactions which traverse the protein that act asthe allosteric communication channel between domains (23–25).Elucidating these sectors has advanced our understanding ofhow distinct domains "talk" to one another and has permitteddirect engineering of allosteric responses into non-allostericenzymes (26–28). Even so, we are left without a quantitativeunderstanding of how these admittedly complex networks setthe energetic difference between active and inactive states orhow a given mutation influences binding affinity. In this con-text, a biophysical model in which the various parameters areintimately connected to the molecular details can be of use andcan lead to quantitative predictions of the interplay betweenamino-acid identity and system-level response.

By considering how each parameter contributes to the ob-served change in free energy, we are able to tease out differentclasses of parameter perturbations which result in stereotypedresponses to changing inducer concentration. These characteris-tic changes to the free energy can be used as a diagnostic tool toclassify mutational effects. For example, we show in Fig. 2 thatmodulating the inducer binding constants KA and KI resultsin non-monotonic free energy changes that are dependent onthe inducer concentration, a feature observed in the inducerbinding mutants examined in this work. Simply looking atthe inferred DF as a function of inducer concentration, whichrequires no fitting of the biophysical parameters, indicates thatKA and KI must be modified considering those are the onlyparameters which can generate such a response.

Another key observation is that a perturbation to only KAand KI requires that the DF = 0 at c = 0. Deviations from this

condition imply that more than the inducer binding constantsmust have changed. If this shift in DF off of 0 at c = 0 is notconstant across all inducer concentrations, we can surmise thatthe energy difference between the allosteric states D#AI mustalso be modified. We again see this effect for all of our inducermutants. By examining the inferred DF, we can immediatelysay that in addition to KA and KI , D#AI must decrease rela-tive to the wild-type value as DF > 0 at c = 0. When theallosteric parameters are fit to the induction profiles, we indeedsee that this is the case, with all four mutations decreasing theenergy gap between the active and inactive states. Two of thesemutations, Q294R and Q294K, make the inactive state of therepressor more stable than the active state, which is not the casefor the wild-type repressor (10).

Our formulation of DF indicates that shifts away from 0that are independent of the inducer concentration can onlyarise from changes to the repressor copy number and/or DNAbinding specificity, indicating that the allosteric parametersare untouched. We see that for three mutations in the DNAbinding domain, DF is the same irrespective of the inducerconcentration. Measurements of DF for these mutants withrepressor copy numbers across three orders of magnitude yieldapproximately the same value, revealing that D#RA is the soleparameter altered via the mutations.

We note that the conclusions stated above can be qualita-tively drawn without resorting to fitting various parametersand measuring the goodness-of-fit. Rather, the distinct behaviorof DF is sufficient to determine which parameters are changing.Here, these conclusions are quantitatively confirmed by fittingthese parameters to the induction profile, which results in ac-curate predictions of the fold-change and DF for nearly everystrain across different mutations, repressor copy numbers, andoperator sequence, all at different inducer concentrations. With

8

a collection of evidence as to what parameters are changingfor single mutations, we put our model to the test and drewpredictions of how double mutants would behave both in termsof the titration curve and free energy profile.

A hypothesis that arises from our formulation of DF is thata simple summation of the energetic contribution of each mu-tation should be sufficient to predict the double mutants (solong as they are in separate domains). We find that such a cal-culation permits precise and accurate predictions of the doublemutant phenotypes, indicating that there are no epistatic inter-actions between the mutations examined in this work. Withan expectation of what the free energy differences should be,epistatic interactions could be understood by looking at howthe measurements deviate from the prediction. For example, ifepistatic interactions exist which appear as a systematic shiftfrom the predicted DF independent of inducer concentration,one could conclude that DNA binding energy is not equal tothat of the single mutation in the DNA binding domain alone.Similarly, systematic shifts that are dependent on the inducerconcentration (i.e. not constant) indicate that the allosteric pa-rameters must be influenced. If the expected difference in freeenergy is equal to 0 when c = 0, one could surmise that themodified parameter must not be D#AI nor D#RA as these wouldboth result in a shift in leakiness, indicating that KA and KI arefurther modified.

Ultimately, we present this work as a proof-of-principle forusing biophysical models to investigate how mutations influ-ence the response of allosteric systems. We emphasize that sucha treatment allows one to boil down the complex phenotypicresponses of these systems to a single-parameter descriptionwhich is easily interpretable as a free energy. The general utilityof this approach is illustrated in Fig. 6 where gene expres-sion data from previous work (4, 6, 10) along with all of themeasurements presented in this work collapse onto the mastercurve defined by Eq. (3). While our model coarse grains manyof the intricate details of transcriptional regulation into twostates (one in which the repressor is bound to the promoter andone where it is not), it is sufficient to describe a wide rangeof regulatory scenarios. Given enough parametric knowledgeof the system, it becomes possible to examine how modifica-tions to the parameters move the physiological response alongthis reduced one-dimensional parameter space. This approachoffers a glimpse at how mutational effects can be describedin terms of energy rather than Hill coefficients and arbitraryprefactors. While we have explored a very small region of se-quence space in this work, coupling of this approach with high-throughput sequencing-based methods to query a library ofmutations within the protein will shed light on the phenotypiclandscape centered at the wild-type sequence. Furthermore,pairing libraries of protein and operator sequence mutants willprovide insight as to how the protein and regulatory sequencecoevolve, a topic rich with opportunity for a dialogue betweentheory and experiment.

Materials and Methods

Bacterial Strains and DNA Constructs. All wild-type strains fromwhich the mutants were derived were generated in previous workfrom the Phillips group (4, 10). Briefly, mutations were first introducedinto the lacI gene of our pZS3*1-lacI plasmid (4) using a combinationof overhang PCR Gibson assembly as well as QuickChange mutagen-esis (Agligent Technologies). The oligonucleotide sequences used to

Fig. 6. Data collapse of the simple repression regulatory architecture. All data aremeans of biological replicates. Where present, error bars correspond to the standarderror of the mean of five to fifteen biological replicates. Red triangles indicate datafrom Garcia and Phillips (4) obtained by colorimetric assays. Blue squares are datafrom Brewster et al.(6) acquired from video microscopy. Green circles are data fromRazo-Mejia et al. (10) obtained via flow cytometry. All other symbols correspond tothe work presented here. An interactive version of this figure can be found on thepaper website where the different data sets can be viewed in more detail.

generate each mutant as well as the method are provided in the SI text.For mutants generated through overhang PCR and Gibson assembly,

oligonucleotide primers were purchased containing an overhang withthe desired mutation and used to amplify the entire plasmid. Using thehomology of the primer overhang, Gibson assembly was performed tocircularize the DNA prior to electroporation into MG1655 E. coli cells.Integration of LacI mutants was performed with l Red recombineering(29) as described in Reference (4).

The mutants studied in this work were chosen from data reportedin (5). In selecting mutations, we looked for mutants which suggestedmoderate to strong deviations from the behavior of the wild-type re-pressor. We note that the variant of LacI used in this work has anadditional three amino acids (Met-Val-Asn) added to the N-terminusthan the canonical LacI sequence reported in (30). For this reason, allmutants given here are with respect to our sequence and their positionsare shifted by three to those studied in (5).

Flow Cytometry. All fold-change measurements were performed ona MACSQuant flow cytometer as described in Razo-Mejia et al. (10).Briefly, saturated overnight cultures 500 µL in volume were grown indeep-well 96 well plates covered with a breathable nylon cover (LabPak - Nitex Nylon, Sefar America, Cat. No. 241205). After approxi-mately 12 to 15 hr, the cultures reached saturation and were diluted1000-fold into a second 2 mL 96-deep-well plate where each well con-tained 500 µL of M9 minimal media supplemented with 0.5% w/vglucose (anhydrous D-Glucose, Macron Chemicals) and the appro-priate concentration of IPTG (Isopropyl b-D-1-thiogalactopyranoside,Dioxane Free, Research Products International). These were sealedwith a breathable cover and were allowed to grow for approximately8 hours until the OD600nm ⇡ 0.3. Cells were then diluted ten-fold intoa round-bottom 96-well plate (Corning Cat. No. 3365) containing 90µL of M9 minimal media supplemented with 0.5% w/v glucose alongwith the corresponding IPTG concentrations.

The flow cytometer was calibrated prior to use with MACSQuantCalibration Beads (Cat. No. 130-093-607). During measurement, thecultures were held at approximately 4� C by placing the 96-well plateon a MACSQuant ice block. All fluorescence measurements were madeusing a 488 nm excitation wavelength with a 525/50 nm emission filter.The photomultiplier tube voltage settings for the instrument are thesame as those used in Reference (10).

The data was processed using an automatic unsupervised gating

9

https://www.rpgroup.caltech.edu/mwc_mutants

procedure based on the front and side- scattering values, where we fita two-dimensional Gaussian function to the log10 forward-scattering(FSC) and the log10 side-scattering (SSC) data. Here we assume that theregion with highest density of points in these two channels correspondsto single-cell measurements and consider data points that fall within40% of the highest density region of the two-dimensional Gaussianfunction. We direct the reader to Reference (10) for further detail andcomparison of flow cytometry with single-cell microscopy.

Bayesian Parameter Estimation. We used a Bayesian definition of prob-ability in the statistical analysis of all mutants in this work. In the SItext, we derive in detail the statistical models used for the variousparameters as well as multiple diagnostic tests. Here, we give a genericdescription of our approach. To be succinct in notation, we considera generic parameter q which represents D#RA, KA, KI , and/or D#AIdepending on the specific LacI mutant.

As prescribed by Bayes’ theorem, we are interested in the posteriorprobability distribution

g(q | y) µ f (y | q)g(q), [7]

where we use g and f to represent probability densities over param-eters and data, respectively, and y to represent a set of fold-changemeasurements. The likelihood of observing our dataset y given a valueof q is captured by f (y | q). All prior information we have about thepossible values of q are described by g(q).

In all inferential models used in this work, we assumed that allexperimental measurements at a given inducer concentration werenormally distributed about a mean value µ dictated by Eq. (1) with avariance s2,

f (y | q) = 1(2ps2)N/2

N

’i

exp� (yi � µ(q))

2

2s2

�, [8]

where N is the number of measurements in the data set y.This choice of likelihood is justified as each individual measure-

ment at a given inducer concentration is a biological replicate andindependent of all other experiments. By using a Gaussian likelihood,we introduce another parameter s. As s must be positive and greaterthan zero, we define as a prior distribution a half-normal distributionwith a standard deviation f,

g(s) =1f

r2p

exp� x

2f2

�; x � 0, [9]

where x is a given range of values for s. A standard deviation of f = 0.1was chosen given our knowledge of the scale of our measurementerror from other experiments. As the absolute measurement of fold-change is restricted between 0 and 1.0, and given our knowledge ofthe sensitivity of the experiment, it is reasonable to assume that theerror will be closer to 0 than to 1.0. Further justification of this choiceof prior through simulation based methods are given in the SI text.The prior distribution for q is dependent on the parameter and itsassociated physical and physiological restrictions. Detailed discussionof our chosen prior distributions for each model can also be found inthe SI text.

All statistical modeling and parameter inference was performedusing Markov chain Monte Carlo (MCMC). Specifically, HamiltonianMonte Carlo sampling was used as sis implemented in the Stan prob-abilistic programming language (31). All statistical models saved as.stan models and can be accessed at the GitHub repository associatedwith this work (DOI: 10.5281/zenodo.2721798) or can be downloadeddirectly from the paper website.

Inference of Free Energy From Fold-Change Data. While the fold-change in gene expression is restricted to be between 0 and 1, exper-imental noise can generate fold-change measurements beyond thesebounds. To determine the free energy for a given set of fold-changemeasurements (for one unique strain at a single inducer concentration),we modeled the observed fold-change measurements as being drawnfrom a normal distribution with a mean µ and standard deviation s.Using Bayes’ theorem, we can write the posterior distribution as

g(µ, s |y) µ g(µ)g(s) 1(2ps2)N/2

N

’i

exp�(yi � µ)2

2s2

�, [10]

where y is a collection of fold-change measurements. The prior distri-bution for µ was chosen to be uniform between 0 and 1 while the prioron s was chosen to be half normal, as written in Eq. (9). The posteriordistribution was sampled independently for each set of fold-changemeasurements using MCMC. The .stan model for this inference isavailable on the paper website.

For each MCMC sample of µ, the free energy was calculated as

F = � log⇣

µ�1 � 1⌘

[11]

which is simply the rearrangement of Eq. (3). Using simulated data, wedetermined that when µ < s or (1 � µ) < s, the mean fold-change ingene expression was over or underestimated for the lower and upperlimit, respectively. This means that there are maximum and minimumlevels of fold-change that can be detected using flow cytometry whichare set by the distribution of fold-change measurements resulting fromvarious sources of day-to-day variation. This results in a systematicerror in the calculation of the free energy, making proper inferencebeyond these limits difficult. This bounds the range in which we canconfidently infer this quantity with flow cytometry. We hypothesizethat more sensitive methods, such as single cell microscopy, colorimet-ric assays, or direct counting of mRNA transcripts via Fluorescence InSitu Hybridization (FISH) would improve the measurement of DF. Wefurther discuss details of this limitation in the SI text.

Data and Code Availability. All data was collected, stored, and pre-served using the Git version control software. Code for data processing,analysis, and figure generation is available on the GitHub repository(https://www.github.com/rpgroup-pboc/mwc_mutants) or can beaccessed via the paper website. Raw flow cytometry data is storedon the CaltechDATA data repository and can be accessed via DOI10.22002/D1.1241.

ACKNOWLEDGMENTS. We thank Pamela Björkman, Rachel Gal-imidi, and Priyanthi Gnanapragasam for access and training for the useof the Miltenyi Biotec MACSQuant flow cytometer. The experimentalefforts first took place at the Physiology summer course at the MarineBiological Laboratory in Woods Hole, MA, operated by the Univer-sity of Chicago. We thank Ambika Nadkarni and Damian Dudka fortheir work on the project during the course. We also thank SuzannahBeeler, Justin Bois, Robert Brewster, Soichi Hirokawa, Heun Jin Lee,and Muir Morrison for thoughtful advice and discussion. This workwas supported by La Fondation Pierre-Gilles de Gennes, the RosenCenter at Caltech, the NIH DP1 OD0002179 (Director’s Pioneer Award),R01 GM085286, and 1R35 GM118043 (MIRA). Nathan M. Belliveauwas supported by a Howard Hughes Medical Institute InternationalStudent Research fellowship.

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Materials and Methods