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Cell, Volume 139

Supplemental Data

Transient Non-native HydrogenBonds Promote Activation

of a Signaling Protein

Alexandra K. Gardino, Janice Villali, Aleksandr Kivenson, Ming Lei, Ce Feng Liu, Phillip

Steindel, Elan Z. Eisenmesser, Wladimir Labeikovsky, Magnus Wolf-Watz, Michael W.

Clarkson, and Dorothee Kern

Figure S1:15 N CPMG relaxation dispersion for wild-type (A), BeF3

--activated (B) S85D

(C), and Y101F NtrCr (D) at 800 MHz and 25C. The dispersion curves are color coded as

followed: residue 5 (peach), 8 (gold), 10 (burnt sienna), 11 (black), 12 (slate), 13

(strawberry), 16 (rose), 29 (salmon), 37 (burgandy), 40 (dark gray), 49 (sage), 50

(mustard), 55 (yellow-green), 64 (brown), 66 (blue-green), 69 (red), 71 (light gray), 88

(orange), 89 (dark green), 90 (light brown), 91 (navy blue), 102 (royal blue), 106 (sea

green), 107 (jungle green), 114 (tan), 122 (dark brown).

Global fitting of the 600 MHz and 800 MHz CPMG data for the BeF 3-activated form and

the transition-state mutants (S85D and Y101F) confirmed both the exchange rates and the

populations previously determined using the chemical shift changes of all mutant forms

together with the CPMG experiments at 600 MHz only, therefore independently verifying

the robustness of determining these kinetic parameters using the approach described in

Experimental Procedures. For wild-type, such a global fit is not possible because the

exchange rate is too fast to be fully suppressed by the maximal CPMG field strength.

Here, exchange rates were determined according to (Gardino and Kern, 2007), and

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populations as described in Experimental Procedures. The CPMG data at 800 MHz are

consistent with the inactive/active conformational exchange occurring in the very fast

NMR time regime (k ex>> Δω), in which R ex is proportional to Δω2. Error in relaxation

rates are from the larger of the difference in duplicate points or 2% of the signal to noise.

Figure S2. Kinetic and thermodynamic data for folding of inactive and active wild-

type and mutant forms of NtrCr

measured by fluorescence.

(A) Chevron plot depicting the natural log of the observed rate (k obs), where k obs is equal

to the sum of the unfolding rate and folding rate (k obs= k u + k f ) for WT (red▲

), D86N

(orange ■), D86N/A89T (yellow ♦), and CPO4-activated NtrCr

(black ●). Rates were

measured using stopped-flow fluorescence at various guanidinium-hydrochloride

concentrations at 25°C. Error in unfolding rates are s.d. from the mean. (B)

Thermodynamic unfolding curve for wild-type NtrCr

measured by fluorescence after

equilibration for 1 hour at 25°C at various GdmCl concentrations. Error in fluorescence

unfolding data are s.d. from the mean. (C) Kinetic scheme illustrating why the

carbamoylphosphate-activated NtrCr

shows the characteristic U-shape of a chevron plot

in the region of the folding transition while wild-type, D86N, and D86N/A89T mutants

do not. Rates for proline isomerization at 25°C were estimated from previously

published data (Reimer et al., 1998; Schutkowski et al., 1994). According to (B) for

wild-type NtrCr , k obs should curve below 3.3 M GdmCl due to the increasing contribution

of k f with an inflection point at 2.5M (k u = k f ). We propose that the lack of curvature in

the transition region of the Chevron plot is due to the cis/trans isomerization of the Pro

105 cis-proline in the structure of NtrCr

after unfolding. For wild-type and mutants

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forms, the cis/trans isomerization is at the same magnitude as the refolding rate resulting

in the lack of curvature in the Chevron plot since there is no significant refolding because

the majority of molecules are in the wrong isomerization state (trans). In contrast, for P-

the cis/trans isomerization is one order of magnitude faster than the refolding rate

consequently is no longer rate limiting. This results in the typical curving of the Chevron

plot for the latter. This model is buttressed by our stopped flow refolding experiments.

When refolding was initiated by dilution of the denaturant after incubation, a double

exponential process as detected with a fast rate followed by a slow rate of about 10-2

the time regime indicative for prolyl peptide bond isomerization. The second slow

process was eliminated in a double jump experiment in which the protein was only

allowed to unfold for 10 seconds (Kiefhaber and Schmid, 1992). Folding rates plotted

with open symbols in (A) are from the double jump experiments. We note that a very

similar model was used in folding studies of the homologous protein CheY (Munoz et al.,

1994). Rates of folding and unfolding determined from the chevron plot are listed in

Table S2.

Figure S3. Quantitative fits of the folding/unfolding equilibrium measured by

(A) Change in peak intensity for W7 (peak of the folded state in a15 N

1H HSQC) in wild-

type NtrCr

with increasing denaturant concentration. (B) Effect of increasing

concentrations of salt (NaCl) on the peak intensity of W7 follows a single exponential.

(C) Change in intensity with increasing GdmHCl (same data as in a) fit to a modified

sigmoidal characterized by a two-state transition centered ~2.4M GdmHCl,

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corresponding to the unfolding transition, and an exponential to account for the change in

intensity due to an increase in ionic strength of the solution. (D) Plot of peak intensity

after subtraction of the ionic strength effect which can now be fitted to a standard two-

state sigmoidal. Error from the r.m.s.d. from the mean over a 1ppm (1H dimension) by

3ppm (15N dimension) in a peak free region of the individual spectra to estimate spectral

noise.

Figure S4. Residues that were fit as described in Fig. S3 to determine their change in

free energy of unfolding (Δ

GUF) are plotted onto the inactive state structure in red.

Grey residues are unassigned, prolines, or overlapped. Residues in yellow are assigned

peaks whose intensities were small due to exchange broadening. This lead to a complete

loss of the peak intensity at small concentrations of denaturant due to the effect of

increasing ionic strength of the sample, and thus no information on the unfolding/folding

transition.

Figure S5. The change in free energy of unfolding (ΔGUF, A) and m-value (MG, B)

for each residue in NtrCr

fitted as described in Fig. S3.

The intensity of each peak in the absence of denaturant (C) showing that residues with

large errors in ΔGUF (A) and MG (B) correspond to residues with lower peak intensities

due to exchange broadening. Error derived from the r.m.s.d. from the mean over a 1ppm

(1H dimension) by 3ppm (15N dimension) in a peak free region of the individual spectra

to estimate spectral noise.

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Figure S6. S85D mutation that removes a non-native hydrogen bond of the

transition does not affect the ground states (inactive and active state).

(A) Overlay of 1H-

15 N HSQC spectra of NtrC

r wild-type (red) and S85D (blue) at 25°C

indicating that the structures and the active/inactive equilibrium are not altered by this

mutation. (B) The unfolding rates (see also Fig. S 2) of wild-type (red ) and S85D

(blue ) are identical within experimental error. Rates were measured using stopped-

flow fluorescence at various guanidinium-hydrochloride concentrations at 25°C. Data

were linearly extrapolated to determine the rates of unfolding in the absence of

denaturant for each NtrCr

mutant, which are listed in Table S3. Error in unfolding rates

are s.d. from the mean.

Figure S7. Unbiased MD simulations in explicit water for wild-type, S85D and

S85G support the experimental finding that the ground states are not significantly

changed by these amino acid substitutions.

(A) Top panel: Time traces of backbone root mean square deviation (RMSD) along the

MD trajectories of the inactive (blue) and active (red) states. (B) Bottom panel: The

backbone root mean square fluctuation (RMSF) of the simulation trajectories between 9 -

15 ns are plotted for wild-type inactive (thick blue line) and active (thick red line), S85D

inactive (darkest blue) and active (brown) and S85G inactive (light blue) and active

(gold). The only small noticeable change is an increased RMSF for helix 4 for the S85G

mutation for the active state most likely due to the introduction of a glycine residue.

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Figure S8. Mutation identified to be involved in the pathway by TMD (Lei et al.,

2009) that is not rate-limiting for the activation process.

15 N CPMG NMR relaxation dispersion data (Palmer et al., 2001) for G97A NtrC

indicating that the rate of inactive/active interconversion is similar to the rate for the

wild-type form. The dispersion curves are color coded as followed: residue 6 (ivory), 9

(raspberry), 11 (black), 29 (salmon), 30 (plum), 35 (lavender), 50 (mustard), 78 (cyan),

82 (magenta), 91 (navy blue), 102 (royal blue), and 122 (dark brown).

In the TMD

trajectory (Lei et al., 2009), G97 samples phi and psi angles that are only in allowed

regions in the Ramachandran plot for a glycine residue. We wanted to test the effect of

an alanine mutation in this position on the overall rate of inactive/active interconversion.

Apparently, this mutation does not alter the overall rate, although it is possible that the

energy barrier of this step in the transition pathway is increased but it must be still lower

than the highest energy barrier. This mutation serves a negative control, a mutation that

does not alter the macroscopically observed kinetics.

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0 800600400200 1000

0 200 400 600 800 1000

8006004002000 1000

1000800600400200

e f f ( s

CPMG (Hz) CPMG (Hz)

Figure S1

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0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6

GdmHCl ([M])

Utrans Ucis Fcis

W e i g h t e d A v g . F l u o r e s c e n c e

E m i s s i o n f r o m 3 0 0 - 4 5 0 ( n m )

l n ( k ) , (

s ) - 1

←→ →

→→

← ←

WT, Mut

P-NtrC

s-1 s-1

s-1s-1

Figure S2

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P e a k

I n t e n s i t y

P e a k

I n t e n s i t y

P e a k

I n t e n s i t y

[GdmHCl], M

[NaCl], M

[GdmHCl], M

Figure S3

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Figure S4

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80 100 1206040200

Residue

100806040200

12010080604020

( k c a l / m o l )

I n t e n s i t y

Figure S5

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10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0130

1 5 N ( p p m )

1H (ppm)

2.5 3 3.5 4

l n ( k

GdmHCl (M)

Figure S6

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Figure S7

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0 200 400 600 800 1000

e f f ( s

CPMG (Hz) ν

Figure S8

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Table S1 – Chemical Shift Differences Between the Inactive (ωI) and Active

(ωA) States Calculated From Relaxation Dispersion Data and theCorresponding Populations

________________________________________________________________

Δω|ωI - ωA| p.p.m.

________________________________________________________________

residue WT D86N D86NA89T BeF3-

pA 0.14±0.02 0.43±0.03 0.65±0.05 0.995±0.002

11 1.64±0.4 1.03±0.3 1.39±0.3 1.79±0.569 1.30±0.4 1.62±0.270 1.32±0.3 1.08±0.372 1.07±0.2 1.08±0.278 2.38±0.7 1.32±0.482 2.35±0.2 2.21±0.287 1.33±0.2 1.08±0.15 1.22±0.188 2.45±0.6 1.75±0.3 2.24±0.4 2.18±0.389 2.48±0.25 2.39±0.291 1.75±0.4 1.29±0.2

100 1.64±0.5 1.67±0.45 2.39±0.55

* Chemical shifts are reported for those residues in NtrC that hadquantifiable chemical exchange when fit to the Carver-Richards equation.Missing chemical shift values are indicative of residues that are severelyexchange broadened or overlapped. All errors are one s.d.

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Table S2. Folding and unfolding rates and comparison of the free energy ofactivation of unfolding to the corresponding free energy change caused by

constitutively activating mutations or phosphorylation

kf (s-1)

ku (s-1)

ΔΔG‡ of unfolding

compared to wt

(cal/mol)a

ΔΔG comparto wt from

CPMG data

(cal/mol)b

WT 51.9 ± 7.87.66E-06 ±1.61E-06

D86N 49.4 ± 8.42.32E-05 ±3.72E-06

600 ± 300 700 ± 60

D86N/A89T 48.9 ± 12.22.90E-05 ±

1.22E-05

700 ± 400 750 ± 70

Activated (C-PO4) -3.36E-08 ±1.17E-08

-3300 ± 400 -3240 ± 320

a) ku are used in the Eyring equation for the calculation, see also methods.b) Data used here, see Figure 2E.All errors are one s.d.

Table 3. Unfolding rates and correspondingfree energy of activation of unfolding for

mutant forms that effect the transition landscape

ku (s-1)

ΔΔG‡ of unfolding

compared to wt

(cal/mol) a

WT7.66E-06 ±1.61E-06

S85D8.05E-06 ±2.17E-06

0 ± 400

a) ku are used in the Eyring equation for the

calculation, see also methodsAll errors are one s.d.

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Supplemental References

Gardino, A.K., and Kern, D. (2007). Functional dynamics of response regulators using

NMR relaxation techniques. Methods Enzymol 423, 149-165.

Kiefhaber, T., and Schmid, F.X. (1992). Kinetic coupling between protein folding and

prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1. J Mol Biol 224,

231-240.

Lei, M., Velos, J., Gardino, A., Kivenson, A., Karplus, M., and Kern, D. (2009).

Segmented Transition Pathway of the Signaling Protein Nitrogen Regulatory Protein C. J

Mol Biol.

Munoz, V., Lopez, E.M., Jager, M., and Serrano, L. (1994). Kinetic characterization of

the chemotactic protein from Escherichia coli, CheY. Kinetic analysis of the inverse

hydrophobic effect. Biochemistry 33, 5858-5866.

Palmer, A.G., 3rd, Kroenke, C.D., and Loria, J.P. (2001). Nuclear magnetic resonance

methods for quantifying microsecond-to-millisecond motions in biological

macromolecules. Methods Enzymol 339, 204-238.

Reimer, U., Scherer, G., Drewello, M., Kruber, S., Schutkowski, M., and Fischer, G.

(1998). Side-chain effects on peptidyl-prolyl cis/trans isomerisation. J Mol Biol 279, 449-

Schutkowski, M., Neubert, K., and Fischer, G. (1994). Influence on proline-specific

enzymes of a substrate containing the thioxoaminoacyl-prolyl peptide bond. Eur J

Biochem 221, 455-461.

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