A Double Resonance CEST Experiment to Study Multistate Protein

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

A Double Resonance CEST Experiment to Study Multistate Protein Conformational Exchange: An Application to Protein Folding Pramodh Vallurupalli, Ved Prakash Tiwari, and Shamasree Ghosh J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b00985 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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The Journal of Physical Chemistry Letters

A Double Resonance CEST Experiment to Study Multistate Protein Conformational Exchange: An Application to Protein Folding

Pramodh Vallurupalli*, Ved Prakash Tiwari and Shamasree Ghosh

TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/P, Gopanpally Village, Serilingampally Mandal, Ranga Reddy District, Hyderabad, Telangana 500107, India E-mail: [email protected] Corresponding Author * [email protected]

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Abstract Despite the importance of protein dynamics to function, studying exchange between multiple conformational states remains a challenge because sparsely populated states are invisible to conventional techniques. CEST NMR experiments can detect minor states with lifetimes between 5 and 200 milliseconds populated to just 1%. However CEST often cannot provide the exchange mechanism for processes involving three or more states leaving the role of the detected minor states unknown. Here a double resonance CEST experiment to determine the kinetics of multistate exchange is presented. The approach that involves irradiating resonances from two minor states simultaneously is used to study the exchange of T4 lysozyme (T4L) between the dominant native state and two minor states, the unfolded state and a second minor state (B), each populated to only ~4%. Regular CEST does not provide the folding mechanism but double resonance CEST clearly shows that T4L can fold directly without going through B.

TOC GRAPHICS

KEYWORDS Protein Conformational Dynamics, NMR, Chemical Exchange Saturation Transfer, CEST, Chemical Exchange, Invisible States


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It is now well established that protein molecules are dynamic adopting different conformations in solution.1-4 Knowledge of this dynamics can be crucial to understanding protein function1-2, 5-6 and necessary to understand processes like protein folding, misfolding and aggregation.1, 7-8 Exchange often occurs between a highly populated (‘visible’) major state that is detected by conventional biophysical techniques and sparsely populated (‘invisible’) minor state(s) that cannot be detected by traditional techniques and methods are actively being developed to detect these states.1, 9-10 Principle among these are NMR relaxation experiments that can detect minor conformers populated to just 1% with lifetimes varying from ~5 μs to 500 milliseconds (ms).1,

9

These

experiments probe the dynamics at several backbone and sidechain sites in each residue of the protein resulting in atomic resolution models of the minor conformers in favorable circumstances.11-14 The CEST and related DEST class of NMR experiments that can detect protein minor conformers with lifetimes in the ~5 to 200 ms range and populations as low as 1%15-18 are routinely used to study various exchange processes related to protein function,19-22 ligand binding,23-26 folding27 and aggregation.16, 28 In a traditional CEST experiment the intensity of the visible major state peak is monitored as a function of the offset frequency at which a weak “saturating” transverse B1 field is applied for a time TEX (~200–600 ms).17, 29-30 Magnetization is along z at the start of TEX and z magnetization at the end of the TEX delay is detected. When the offset frequency matches a minor state resonance frequency, spins in that state precess around the B1 field and are not along z when exchange returns them to the major state, resulting in a loss in major state intensity. A plot of major state intensity vs. offset shows dips at the resonance frequencies of the (major and) minor state(s) that are in exchange with the major state directly or indirectly allowing one to reconstruct the spectrum of the minor state(s). Analysis of CEST data recorded at a few B1 values can provide the exchange rate(s) and populations in addition to the


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“spectra” of the minor state(s). Although CEST experiments are used to detect and study exchange between three or more states, often detailed kinetics cannot be extracted from CEST data for multistate exchange9, 27 leaving the role of the detected minor states in the process being studied unknown. For example in folding studies it may not be possible to distinguish onpathway folding intermediates from off-pathway minor conformers and developing methods to study multistate exchange is an active area of research.9,

31-32

Herein we describe a double

resonance CEST experiment to study multistate exchange and demonstrate its utility by using it to study the three state conformational exchange of T4 lysozyme (T4L) between a dominant native folded form (F) and two minor states (B and U). T4L has been used extensively to study protein plasticity, dynamics and folding.13, 33-43 The backbone amide 15N-1H HSQC is well resolved (Figure 1a) and exchange could be studied at 144 sites. Most of the amide 15N CEST profiles recorded at 50 oC show at least one dip arising from a minor state while two minor state dips are present in the intensity profiles of many residues from the N terminal domain (Figure 1b) showing that exchange occurs between F and at least two minor states. CEST data was acquired using the D-CEST sequence44 that accelerates data acquisition by using the DANTE scheme45 to perform multifrequency irradiation at a desired effective B1 (B1,eff) field. Global three state exchange models (See SI) where the exchange rates (𝑘!"#$ , 𝑘!"#$ , 𝑘!"#$ ) and fractional populations ( 𝑝! , 𝑝! ) are the same for all sites (Figure 1b, ! c; S1; S2; Table S1) could fit (reduced 𝜒 ! , 𝜒!"# ~1) amide 15N D-CEST data recorded at three

B1,eff values (1.6, 8.2 and 15.9 Hz, Table S2). Global fits were carried out using data from six residues K19, D20, T26, L32, K35 and L39 but the conclusions remain the same with more residues. Figure 1d shows the variation in the magnitude of the chemical shift differences


4

a 110

Y18 I29

T59 A82

G30 G51 T21

L39

N (ppm)

D89 D127

15

120

S136 T115

R137 F104 L15 V71 L33 9.5

9.0

f

K16 N2 V87

I/I0

A98 W126

L91

0.40

R14 T142 T157 8.5 8.0 7.5

H (ppm)

0.32 0.48

0.32

L164

1

c

b D-CEST B1,eff = 8.2 Hz SWD = 900 Hz

T26

T26

D-CEST B1,eff = 15.9 Hz SWD = 1250 Hz ϖB

0.32

I/I0

5

12

|ϖU-ϖF| |ϖB-ϖF|

117.5

ϖU ϖF

T26

120.0

D-CEST B1,eff = 15.9 Hz ϖ SWD = 1250 Hz B

122.5 115.0

117.5

ϖF 120.0

122.5

8 6 4 2 0 0

0.48 D-CEST B1,eff = 8.2 Hz SWD = 900 Hz

0.40 10

D-CEST B1,eff = 8.2 Hz SWD = 900 Hz

T26

ϖU 115.0

7.0

d

10

0.40

K135

T26 I150 N55 K60

10.0

0.48

N140

E11

CEST derived ϖU-ϖF (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CEST derived |ϖU-ϖF| |ϖB-ϖF| (ppm)

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L32

40

60

80

100 120 140 160

Residue Number

e


L32

20

0.48

0

0.40 0.32

-5

ϖU L32


124.0

-10

RMSD 1.7 ppm -10

-5

0

5

10

Predicted ϖUnfolded-ϖF (ppm)

kex pB/U

F

ϖB

128.0

B

18.1 s-1

5.8%

L32

132.0

ϖ (ppm) 7.4 s-1

ϖU ϖF

U

5.4%


124.0

kex pB/U

128.0

4.3%

F

7.2 s-1

ϖF 132.0

ϖ (ppm)

10.5 s-1

B

ϖB

U

3.1%

--Figure 1. CEST detects two minor conformers of T4L. a) Amide HSQC of T4L at 50 oC. b,c) D-CEST profiles of T26 and L32. Global fits to the F⟷B⟷U model (b, purple) and the B⟷F⟷U model (c, green) carried out using D-CEST data for six residues (Table S1). Experimental data is shown as brown circles with errorbars. For clarity only the 8.2 and 15.9 Hz profiles are shown. d) Variation of |ϖB-ϖF| (blue) and |ϖU-ϖF| (red) along the sequence of T4L shows that conformational differences between B and F are restricted to the stretch from K19 to N40 (grey background). e) Ribbon representation of T4L.46 Residues K19 to N40 are colored in blue, the rest of the N terminal domain (residues 13-18, 41-71) is in orange and the C-terminal domain (residues 1-12, 72-164) is in grey. f) Comparison of the CEST derived ϖU-ϖF values with the difference between predicted unfolded shifts (ϖUnfolded) and ϖF. Residue specific parameters are listed in Table S3.

between B and F |ϖB–ϖF| (blue) and between U and F |ϖU–ϖF| (red) along the sequence. Here ϖi (ppm) is the chemical shift and νi (Hz) is the resonance frequency of a spin in state i. ϖB differs from ϖF in the stretch from K19 to N40 (Figure 1d,e) showing that the conformation of T4L in state B differs from the native form only in this region. Differences between ϖU and ϖF occur all over the sequence (Figure 1d) and the ϖU shifts obtained from the CEST data are in good agreement with those predicted for the unfolded state47 (Figure 1f) showing that T4L is unfolded in U. Despite detecting two minor state conformers, D-CEST data does not provide insights into


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the exchange process exemplified by the fact that the data is consistent with two out of the three linear limiting cases of three state exchange: F⟷B⟷U where folding has to proceed via B ! (𝜒!"# =1) (Figure 1b; S1; Table S1) and B⟷F⟷U where B and U independently interconvert ! with the major state F (𝜒!"# =1) (Figure 1c, S2, Table S1). In what follows F is always the visible ! major state. The D-CEST data rules out (𝜒!"# =2.2) the F⟷U⟷B model in which the molecule

has to unfold to interconvert between F and B (Figure S3; Table S1) probably because 𝑘!"#$ > 𝑘!"#$ . The F⟷B⟷U and B⟷F⟷U models can account for the D-CEST data as profiles calculated using the bestfit exchange parameters for the two models give similar CEST profiles (Purple and green lines are nearly superimposable in Figure 2a). To test if T4L folds directly we carried out double resonance CEST experiments (Figure 2b). Unlike a regular CEST experiment (Figure 2a), in the double resonance CEST experiment simultaneous B1 irradiation is carried out at the two minor state resonance frequencies of the same site (Figure 2b) during TEX and its effect on the visible state magnetization is recorded as in a regular CEST experiment. We note that the size of the dip in a regular CEST profile that occurs at ϖj due to minor state j, depends on the strength of the B1 field, increasing with B1 when 2𝜋𝐵! < 1.25/𝜏! (𝜏! is the lifetime of j).17, 48 When 2𝜋𝐵! > 1.25/𝜏! the spins get almost completely dephased when the molecules ‘visit’ j and the size of the dip begins to plateau with increasing B1.17, 48 In the discussion that follows, for simplicity we assume that |νB- νU|, |νB-νF|, |νU-νF| >> B1 so that B1 irradiation at the resonance frequency of one state will not have an effect on spins in the other two states. When exchange follows the F⟷B⟷U model simultaneous irradiation with 2𝜋𝐵! > 1.25/𝜏! & 1.25/𝜏! at both ϖB and ϖU will not result in a significant additional loss of signal compared to single frequency irradiation at only ϖB (compare purple lines in Figure 2a,b) as the spins would have largely


6

ϖF

kex pB/U kex pB/U

ϖU

0.4

F

7.4 s-1

5.8%

B

4.3%

ϖF

7.2 s-1

F

-5

k

0

ϖ (ppm)

5

0.2

10

5.4%

0.300

U

I/I0

DRD-CEST

3

kexFU (s-1) 30

40

kexFU (s-1)

50

60

0

T26

120.0

118.25

118.5

L32

124.0

128.0

130.0

10

5

10

kexFB (s-1)

15

20

j

130.25 1.6 1.5

6

118.5


ϖB ϖ F

128.0

132.0

DRD-CEST B1,eff = 15.3 Hz SWD = 525.2 Hz

L32

ϖ (ppm)

118.25

124.0

ϖB

129.75

118.0

132.0

122.5

ϖB

L32


L32

f

ϖB

ϖF

120.0


i ϖU


ϖF

117.5

T26

h

U

D-CEST B1,eff = 15.9 Hz SWD = 1250 Hz ϖB

122.5 115.0

8

1 20

ϖU

0.40

l

ϖB ϖF

ϖB

118.0

F

ϖU

e

0.33

5

4

ϖ (ppm)

gB

U


d

0.36

5

10

117.5

0.39 B1,eff = 15 Hz SWD=|νU-νB|

0

B


0.42

ϖB

2

F

T26

0.32

10

1.2

T26

0.48

15

1.6

0

0.32

0.275

20

1.4

ϖU

115.0

DRD-CEST + D-CEST D-CEST

c

0.40

0.325

0.4

B1,eff = 15 Hz SWD=1419 Hz

0.48

3.1%

0.5

ϖB

D-CEST

I/I0

U

0.3

0.2 -10

ϖB

18.1 s-1

B

10.5 s-1

0.3

1.8

ϖU

I

ϖF

0.5

I/I0

b

ϖB

ϖB

129.75

130.0

130.25

ϖ (ppm)

30

m

25

1.4

20

1.3

15

χred2

ϖU

I

pU (%)

a

2 χred

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


φ (%)

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4

1.2

2

1.1

0

1

0

2

4

pB (%)

6

8

10

10 2 χred φ

0

5

10

15

20

5 25

30

0

kexBU (s-1)

Figure 2. Using double resonance D-CEST (DRD-CEST) to study conformational exchange. a) Schematic illustration of the regular CEST experiment. D-CEST profiles calculated with B1,eff =15 Hz for the F⟷B⟷U (purple) and B⟷F⟷U (green) models are nearly superimposable. The exchange parameters (shown) are the bestfit values of the models to the D-CEST data (Table S1) with ϖF =0, ϖB =3.5 and ϖU = -6 ppm. R1 (1.3 s-1) and R2 (7 s-1) were the same for all states, B0 =16.4 T and 3.33 kHz DANTE 15N pulses were applied during TEX. b) Schematic illustration of the DRD-CEST experiment along with profiles calculated for the two models using the same parameters as in (a) but with SWD set to |νB–νU| to satisfy the double resonance condition. The curves are no longer identical suggesting that the DRD-CEST experiment can distinguish between the different exchange models. D & DRD-CEST profiles of T26 (c,d,g,h) and L32 (e,f,i,j). c,d,e,f) Global fits of the F⟷B⟷U (purple) and g,h,i,j) B⟷F⟷U (green) models to ! the D & DRD-CEST data (Table S1). For clarity only the ~15 Hz B1,eff data is shown. k) 𝜒!"# vs kexFU using D-CEST (blue) and D & DRD-CEST data (red). l) Exchange parameters obtained from single residue (red) and global (blue) fits of the triangular model to D & DRD-CEST data. ! m) 𝜒!"# vs. kexBU obtained by analyzing D & DRD-CEST data (red) shows a minimum at kexBU ~0 s-1. The fraction of molecules that fold via B (ϕ= 𝑘!" !

!!"

!"! !!"

𝑘!" + 𝑘!" !

!!"

!"! !!"

)

calculated using the bestfit parameters for different kexBU values (blue).


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dephased when the molecules visited B and additional dephasing when the molecules visit U from B will have a small effect on the detected z magnetization. However when exchange follows the B⟷F⟷U model simultaneous B1 irradiation with 2𝜋𝐵! > 1.25/𝜏! & 1.25/𝜏! will lead to a larger loss in signal than single frequency irradiation at either ϖB or ϖU because B and U exchange independently with F (compare green lines in Figures 2a,b). This can also be understood by examining how the exchange contribution to the major F state relaxation under free precession (𝑅!" ) depends on the rate constants under slow exchange conditions. The three state Bloch-McConnell equations49 for the evolution of transverse magnetization (𝑀! = 𝑀! + 𝑖𝑀! ) components arising from states F, B and U can be written as: 𝑑 𝑀!! 𝑀!! 𝑑𝑡 𝑀 !! −𝑅! − 𝑘!" − 𝑘!" 𝑘!! = 𝑘!"

𝑘!" −𝑅! − 𝑘!" − 𝑘!" + 𝑖Δ𝜔!" 𝑘!"

𝑘!" 𝑘!" −𝑅! − 𝑘!" −𝑘!" + 𝑖Δ𝜔!"

𝑀!! 𝑀!! 𝑀!!

Here 𝑘!" is the rate constant for the i j process and Δ𝜔!" = 2𝜋 𝜈! − 𝜈! . For simplicity the transverse relaxation rate R2 is assumed to be the same for all three states. In the slow exchange limit (𝑘!" + 𝑘!" ≪ |Δ𝜔!" |), we can ignore the off-diagonal terms50 and 𝑅!" = 𝑘!" + 𝑘!" . For the F⟷B⟷U model this reduces to 𝑅!" = 𝑘!" as 𝑘!" = 0, showing that the system can be described as a two state exchange process between F and B.51 However when exchange follows the bifurcated B⟷F⟷U model, 𝑅!" = 𝑘!" + 𝑘!" showing that exchange can be treated as two independent processes F⟷B and F⟷U.51 When Δ𝜔!" = 0 the system can be described by a two state process between “F” and B where U has been subsumed into “F”. In the slow exchange limit the exchange contribution to the relaxation of the dominant peak 𝑅!" Δ𝜔!" = 0 = 𝑘!" for both the F⟷B⟷U and B⟷F⟷U models. Hence if we could compare 𝑅!" measured for the


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three state process Δ𝜔!" ≠ 0 with 𝑅!" measured by setting Δ𝜔!" = 0 (𝑅!" Δ𝜔!" = 0 ), it would be possible to distinguish between the two models. Arbitrarily switching Δ𝜔!" to zero is not possible under free precession conditions but is achieved indirectly by performing double resonance and regular CEST experiments. In a CEST experiment the applied B1 field to some extent plays the role of the B0 field in the free-precession experiment.9, 17 When B1 irradiation is carried out only at ϖB, only spins in B precess around B1 while spins in U and F do not precess around B1, similar to setting Δ𝜔!" =2𝜋𝐵! and Δ𝜔!" =0 in the discussion above but when double resonance irradiation is carried out at ϖB and ϖU spins in B and U precess around B1 while spins in F do not, making it analogous to the three state exchange case discussed above with Δ𝜔!" = Δ𝜔!" = 2𝜋𝐵! . Hence comparing double resonance and regular CEST data (Figure 2a,b) is similar to comparing 𝑅!" Δ𝜔!" ≠ 0 with 𝑅!" Δ𝜔!" = 0 and can distinguish between the linear and bifurcated models. The similarities between regular, double resonance CEST and free precession are limited and several simplifying assumptions were made in the discussion above. Hence the full Bloch-McConnell equations15,

49

for three state exchange without any of the

simplifying assumptions made in the above discussion were used during the fitting process. We should note that when exchange rates are high and the molecules visit the minor state multiple times during TEX, multifrequency irradiation will only have a small additional effect. Additionally if 2𝜋𝐵! < 1.25/𝜏! , ϖB irradiation will only partially dephase the spins in B and simultaneous irradiation at ϖU will lead to additional dephasing when the molecule visits U leading to a larger loss in signal even for the F⟷B⟷U model. Multifrequency CEST irradiation can be performed using a DANTE sequence45 or by using phase or amplitude modulated pulses.50, 52-53 The DANTE sequence that consists of a pulse followed by a 1/SWD delay repeated for the duration of TEX gives rise to narrow excitation bands at ±n×SWD Hz (n=0,1,2,..) from the carrier. Here DANTE


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irradiation using the D-CEST sequence with the carrier at νB and SWD=|νB-νU| was used to perform the double resonance CEST experiment instead of the more general amplitude/phase modulated irradiation as the effect of the DANTE block on the evolution of magnetization during TEX can be evaluated exactly and efficiently.44, 49 The double resonance D-CEST (DRD-CEST) experiment is performed in a site-by-site manner as νB and νU values are site dependent. νB, νU and SWD=|νB-νU| values for different sites were obtained by analyzing D-CEST data. DRDCEST data was recorded for the six sites listed earlier with B1,eff ≈15 Hz (Table S2). Rather than collecting a single datapoint for a νB, νU pair, DRD-CEST experiments were recorded with SWD fixed and the 15N carrier positioned at νB and at offsets of a few Hz around νB (Figures 2b,d,f,h,j; S4; S5; Table S2). Under double resonance conditions discussed above placing the carrier at ϖB is equivalent to placing it at ϖU. In the first saturation transfer study; Forsen and Hoffman used double resonance irradiation on two visible states to study exchange between them.29 When the minor state is visible analyzing minor and major state CEST profiles can disentangle complex kinetics.54 For T4L the size of the dips at ϖB increase upon double resonance irradiation. This becomes apparent for T26 by comparing the dip sizes at ϖB in the D-CEST (Figure 2g) and the DRD-CEST (Figure 2h) profiles and similarly for L32 by comparing the dips in Figures 2i and 2j. Consequently the F⟷B⟷U model does not account for the D & DRD-CEST data (Figures ! 2c,d,e,f; S4) with the bestfit 𝜒!"# increasing to 1.8 from 1 obtained by the analysis of only D-

CEST data (Table S1) showing that folding does not have to proceed via state B. Note that including DRD-CEST data in the fitting process compromises fits to the D-CEST data also for the F⟷B⟷U model (compare 15.9 Hz profiles in Figures S1 & S4). The B⟷F⟷U model ! =1) the D & DRD-CEST data (Figures 2g,h,i,j; S5) with kexFB=10.5±0.5 s-1, accounts for (𝜒!"#


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kexFU=7.1±0.6 s-1, pB=4.3±0.2% and pU=3.2±0.3% showing that T4L can fold directly as ! exemplified in Figure 2k where 𝜒!"# vs. kexFU is plotted for the general three state (triangular)

exchange model where all states interconvert directly with each other (See SI). Analysis of D! CEST data alone does not define kexFU with 𝜒!"# being essentially flat between 0 and ~10 s-1.

Addition of the DRD-CEST data rules out solutions with kexFU ~0 s-1 resulting in a minimum at ~7 s-1. Further, fits of the triangular model to single residue D & DRD-CEST data resulted in similar exchange parameters for all six sites (Figure 2l) supporting the assumption of a global three state process. Single residue as well as global fits to the triangular model resulted in kexBU ! ~0 s-1 (0 – 0.3 s-1 global fits Table S1) and the 𝜒!"# vs. kexBU plot (Figure 2m) shows a minimum

at 0 s-1 suggesting that the F⟷B⟷U path does not have any role in T4L folding. As it is not certain that kexBU =0 and it would not have been surprising if B and U interconverted directly, we calculated ϕ, the fraction of molecules that fold via B (F⟷B⟷U arm of the triangle) for various kexBU values (Figure 2m). When kexBU is set to 1 s-1, the highest value from the monte-carlo trials for uncertainty estimation (See SI) ϕ is ~8% showing that folding via B does not play a ! =1.27) much higher than the 0 major role in T4L folding and even when kexBU is set to 10 s-1 (𝜒!"#

– 0.3 s-1 obtained from the global fits, ϕ is ~26% (Figure 2m) further confirming that the path via B is not the dominant pathway for T4L folding. Native state hydrogen exchange (NSHX) studies have shown that amide protons from residues 12 to 65 exchange faster with solvent suggesting that this region is transiently unfolding35 and forming a folding intermediate.37, 41 As the conformational differences between B and F occur in this region (Figure 1d,e) it is likely that NSHX detected B though the CEST results suggest that B is not a prominent folding intermediate and comparison of ϖB and ϖU shifts suggests that the region from K19 to N40 is not unfolded in B (Figure S6, compare red and blue lines in Figure 1d). These amides may be


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solvent exposed in B or the molecule locally unfolds during the F to B interconversion. An atomic resolution structure of T4L in state B and a better understanding of the folding mechanism will resolve these issues. Attempts are underway to obtain the structure of T4L in state B and to detect other folding intermediates including one suggested by analyzing the unfolded state R2 values (Figure S7). To evaluate the general applicability of the DRD-CEST approach to study three state exchange we generated synthetic data15 using the linear and triangular models, for exchange rates between 10 and 100 s-1 and minor state populations between 1.25 and 5% (See SI). We tested if data generated using the B⟷F⟷U model could be fit using the F⟷B⟷U model and vice versa and if the data generated by the triangular model could be fit with any of the linear models. DCEST data alone often failed to select the right model as wrong models also fit the data. Bestfits to D-CEST data determined the right model in only ~8% of the cases but including DRD-CEST data selects the right model in ~52% of the cases showing the general applicability of the approach (See SI). The DRD-CEST approach can be extended by using different B1 values as in regular CEST and by irradiating different minor state resonances of the same site with different B1 values. Recording data in a multiplexed manner by irradiating minor state pairs from different sites simultaneously can accelerate data acquisition. While studying N (>3) state exchange the double resonance approach can be applied to pairs of resonances from the same site or extended by irradiating up to N-1 minor state resonances from the same site simultaneously. Phase and amplitude modulating B1 rather than DANTE may be more suitable for multisite and multiamplitude irradiation. To conclude we have presented a double resonance CEST approach


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to study multistate exchange in proteins that can provide kinetic parameters not available solely from CEST data. It is expected to play an important role in studying protein multistate exchange.

Experimental Methods NMR experiments were performed on a Bruker Avance III HD 700 MHz spectrometer equipped with a cold probe. Samples contained ~1 mM [U-15N] T4L in 20mM sodium phosphate, 5mM NaN3, 2mM EDTA, 2.5% d6-DMSO, pH 3.0 buffer where d6-DMSO served as the lock.55 (See SI for details) ASSOCIATED CONTENT Supporting Information. Supporting information is available from the ACS website as a single PDF file that contains experimental details, five figures showing fits of various models to the CEST data, a figure showing the variation of |ϖB-ϖF| and |ϖB-ϖU| along the sequence, a figure showing the variation in the R2 of state U (R2U) along the sequence, a table summarizing the results of various best-fits to the CEST data, a table summarizing the different experimental parameters and a table listing the chemical shifts and transverse relaxation rates of different residues in states F, B and U obtained from the analysis of DRD & D-CEST data. AUTHOR INFORMATION Corresponding Author *Email: [email protected]


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ACKNOWLEDGMENTS We thank Dr. T Yuwen & Prof. L E Kay for comments and pulse sequences, Dr. G Bouvignies for the program ChemEx, Dr. A Sekhar for comments, the NMR facility at TIFRH and Dr. K Rao for spectrometer time, TIFR and SERB (ECR/2016/001088) for funds.

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A Double Resonance CEST Experiment to Study Multistate Protein

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