Reliable Tracking In-solution Protein Unfolding via Ultrafast Thermal Unfolding/Ion Mobility-Mass Spectrometry

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1 Supplementary Information Reliable Tracking In-solution Protein Unfolding via Ultrafast Thermal Unfolding/Ion Mobility-Mass Spectrometry,, *,, Department of Chemistry, School of Chemistry and Materials Science and National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui , P. R. China *Corresponding Author: Tel: (+) Fax: (+) Author Contributions G.L. and S.Z. contributed equally. Table of contents: Materials and Methods. Figure S1~S17. Offline and online heating experiments. Circuit design for LHE. Sequential unfolding of human insulin and Aβ (1-40). Sequential unfolding of holo-mb and resultant detachment of heme group. LHE-driven sequential unfolding of CaM in solution. References. 1

2 Materials and Methods. Chemicals. Insulin (recombinant human, min IU/mg, CAS no ) was supplied by SERVA Electrophoresis GmbH (Heidelberg, Baden-Württemberg, Germany). Amyloid β peptide (1-40) [human, Aβ (1-40), CAS no ] was supplied by Abcam Co., Ltd. (Shanghai, China). Myoglobin powder was supplied by Sigma-Aldrich (St. Louis, MO, USA), and other proteins and peptides were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). No further purification was performed for the reagents. All solvents used in this study were of HPLC grade. Purified water (conductivity of 18.2 MΩ.cm) was obtained from a Milli-Q Reference System (Millipore Corp., Bedford, MA, USA). Mass Spectrometer and Ion-Mobility Mass Spectrometry. In typical nanospray experiments, the size of the spray emitter was maintained at ~10 µm. The emitters were pulled from borosilicate glass capillaries using a P-2000 laser-based micropipette puller (Sutter Instruments, Novato, CA, USA). Most experiments were performed using an LTQ-Velos Pro mass spectrometer (Thermo Fisher Scientific, CA, USA). All IMMS data were collected using Waters Synapt G1 instruments. The LTQ-MS operation conditions were as follows (if not otherwise specified): maximum ion injection time 30 ms, and 3 micro-scans for each individual scan. The isolation width was set at 1.0. The MS inlet temperature was 75 C. The Synapt instrument was tuned to allow preservation and transmission of native proteins and protein interactions. This typically involves elevated pressures in the source region (~4 mbar), and decreasing all focusing voltages (e.g., cone, extractor, and bias voltages). The traveling-wave ion mobility separator was operated at a pressure of 3.5 mbar, and DC voltage waves (30 V wave height traveling at 400 m/s) to generate ion mobility separation. The ToF-MS was operated over the m/z range of CCS calibration curves were generated using a previously described protocol, and using literature CCS values derived for use with the Synapt instrument platform. 1-2 Same with previous publication, 1 calibration of travelling-wave IM drift times follows those steps: 1. Prepare calibrant solutions by diluting stocks of melittin, bovine ubiquitin, beta-lactoglobulin and bovine serum albumin in 100 mm ammonium acetate at a concentration of 1-5 μm. 2. Record IM-MS data for ultrafast thermal unfolding proteins at an optimized wave heights and velocity to separate the ions. 3. Use precisely the same instrument conditions (including pressures) for all elements downstream of the trapping ion guide to acquire data for the calibrant proteins. 4. Correct calibrant drift times (acquired using a single wave-height value) for mass-dependent flight time, 2

3 calculated by the equation t D = t D [ c m/z 1000 ], where t D is the corrected drift time in ms, t D is the experimental drift time in ms, m/z is the mass-to-charge ratio of the observed ion and c is a constant. 5. Take calibrant collision cross-sections (Ω) and correct them for both ion charge state and reduced mass (μ) to generate Ω (Ω = Ω/[charge (1/μ) 0.5 ]). 6. Create a plot of ln t D against ln Ω. 7. Fit the plot to a linear relationship of the form: ln Ω = Χ ln t D + ln A, where A is a fit-determined constant and X is referred to as the exponential factor. The correlation coefficient of the fit achieved in this step should be high (R 2 > 0.98). 8. Re-plot Ω versus a new corrected drift time (t D ), where t D is given by [t X D charge (1/μ) 0.5 ]. 9. Use the plot generated in Step 8 to calibrate drift time data for thermal unfolded proteins. LHE configuration and its assembly into nesi-imms. Previously, we developed a variation of nesi, termed as induced nesi (InESI), to extend MS applications to complex biological situations, 3-8 in which electromagnetic induction was attributed to serve as the driving force for electrospray. Here, in addition to serving as an initiator of the electrospray process, electromagnetic induction is known for its heating effect via inductive and capacitive coupling, which is used to perform the thermally induced unfolding (Figure 1). Since the electromagnetic induction occurs within approximately hundreds of milliseconds, its heating effect can be defined within a millisecond level. In a word, the high voltage has two roles in our design: 1) to generate LHE and 2) to initiate nanospray, both of which is followed by IMMS instrument (Waters Synapt G1). LHE was initiated using a homemade power supply system, similar to previous reports. 3-4 The high voltage was originally generated using a 25 MHz function waveform generator (DG1022U, RIGOL, Beijing, China) and was then amplified using a household stereo power amplifier. Then, the generated 100 V p-p pulsed voltage was amplified using a coil up to the final value of 10 kv 0-p. The high voltage was monitored through a digital oscilloscope (DS1052E, RIGOL, Beijing, China) after attenuating (1: 1000) with an attenuation rod. The LHE is regulated through adjusting the amplitude and frequency of this high voltage. Association constant calculation. In studies of low-affinity protein-ligand interactions, which usually use high concentrations of ligands and lead to nonspecific binding during the ESI process, the mathematical treatment of mass spectrometric data was applied to remove the contribution of nonspecific from specific binding

4 The first step involves extracting the nonspecific binding constant from the intensities corresponding to binding numbers that are larger than specific binding numbers, as described below: I 5 I 4 = K n [S] (1) where I n is the intensity with n bound ligands, K n is the value of the nonspecific association binding constant, and [S] is the free ligand concentration. An essential requirement for this method is to study weak complexes because an assumption is made that [S] [S] total. Furthermore, a single binding constant is used by assuming that the proteins are perfectly spherical and the nonspecific binding is independent of each other. Then, the data are corrected to remove the contribution of nonspecific binding, as follows: C N [E] = α (K n [S])i 1 I N (K n [S]) i I N 1 i=1 (2) I 0 where C N is the total concentration of the protein with N ligands bound at specific sites, [E] is the concentration of free protein, and α is the largest number of intensity peaks observed in the spectrum. To obtain the specific binding constant, the following equation is used to relate C N [E] to C N 1 [E] : C N = K [E] N [S] C N 1 [E] (3) where K N is the specific binding constant with the Nth ligand, and the value can be acquired by plotting C N / C N 1 [E] [E] versus [S] for different ligand concentrations. In this case, the intensities of I 4 and I 5 were chosen to extract the nonspecific binding constant by calculating the slope of this linear fitting equation (Eq. 1). To minimize the coefficient of determination, R 2, and to increase the calculation accuracy, the end of the titration curve was used to acquire K n because there was not sufficient nonspecific binding when the ligand concentration was low. Then, the intensity of each peak, I n, the nonspecific binding constant, K n, and the concentration of ligand, [S], can be used to obtain C N /[E] according to Eq. 2. In the final step, the values of the specific binding constant with the Nth ligand, K N, can be determined using Eq. 3. Similarly, the calculations of K 1, K 2, K 3, and K 4 involved different segments of the titration curve to confirm accuracy. For example, we chose a concentration of ligand range from 40 μm to 80 μm to calculate K 4 because the intensity of I 4 was almost invisible when the ligand concentration was lower than 40 μm. Noticeably, each data point was calculated from each independent charge state (6+~14+) in triplicate. It is obvious that the bimodal ion distribution of CaM indicates at least two different conformations, which is consistent with the striking difference in the specific binding constant between CaM 6+~8+ and CaM 9+~14+. 4

5 Figure S1. Snapshot of our setup for the generation of local heating effect (LHE). LHE is generated from the electromagnetic induction heating of a protein solution inside the spray needle, especially at the edge of the needle. To avoid any possible electric shock of the instrument, a ground electrode is mounted at the front of the MS inlet. The voltage for LHE and nanospray is applied to the needle using a coil outside the capillary without direct contact with the sample solution. 5

6 Figure S2. Comparison of LHE-driven Sequential unfolding (a, c) with chemical unfolding (b, d) of cytochrome c. a, b) The difference between voltage-dependent InESI and chemical unfolding was illustrated by comparing the unfolding ratios (16+/8+) of cytochrome c during the voltage variation or HOAc concentration variation. c, d) Typical mass spectra of cytochrome c derived from voltage-dependent LHE and chemical unfolding via HOAc induction are presented. Chemical unfolding exhibited more drastic unfolding processes even under the small amount of HOAc contained in the protein aqueous solution (b, d). The LHE-driven Sequential unfolding of protein is similar to in-spray rapid mixing techniques, which have been increasingly reported for the investigation of the kinetics of protein conformational changes and protein assays. In the chemical unfolding experiments, only limited information (Figure S2d) about the cytochrome c structure was obtained. The voltage-dependent LHE-driven Sequential unfolding results exhibited abundant conformational changes from compactly folded to partially unfolded states, indicated by the shift from a unimodal CSD profile to a bimodal CSD profile (Figure S2a, c) and the corresponding CCS values obtained from IMMS measurements (Figure 2b in the main text). Those observations showed good agreement with previous studies on the acid-induced conformational diversity of cytochrome c

7 Figure S3. The effect of temperature on the CSDs of Mb (10 µm in 1 mm AAT) recorded from nesi-ms (dc 1.5 kv). The temperature of Mb was adjusted by either offline heating of bulk solution (A) or online heating (B) of spray solution in a nesi emitter. Heating was achieved using a dry bath (offline) or an online method in which the spray needle was coiled with a heating ribbon and the temperature was monitored using a calorimeter. The CSD of Mb sprayed from a 63 C solution was significantly broader, and the signal of the protein was severely damaged by overheating. Thus, further heating by elevating the online temperature was prohibited. Offline and online heating experiments. To validate the thermally triggered unfolding assumption, we performed a series of heating experiments including the online and offline heating of Mb solutions. For offline heating, a 50 µl aliquot of Mb (10 µm buffered with 1 mm NH 4 OAc) was individually placed in a dry bath of 95 C for 5 and 15 minutes, respectively. We found that the charge states of heated Mb gradually increased with the increase in offline heating duration (Figure S3a). Furthermore, almost all of the holo-mb turned into apo-mb at 95 C within 15 minutes, which indicated that the gradual unfolding events occurred during the heating of bulk solution. We also conducted online heating experiments to correlate the offline heating-induced unfolding with that of LHE in InESI. The spray emitter was coiled with a heating ribbon to adjust the temperature of the spray solution, and the temperature was monitored using a calorimeter. With an increase in the online heating temperature from 22 C to 50 C (Figure S3b), the dominant charge state of Mb was shifted from 9+ to 10+. In a word, the offline and online temperature-jump experiments have further verified the principle of our strategy. 7

8 Figure S4. Mass spectra of myoglobin (Mb) in 1 mm NH 4 OAc derived from normal nesi-ms without LHE. All results from various dc voltages reported natively folded holo-mb. To correlate the CSD to protein secondary structural changes, we also performed the corresponding CIU-IMMS experiments. As shown in Figure S4, the good relation of measured CCSs with theoretical values from IMPACT calculations 14 and distinctive transitions in CIU profiles 15 strongly supported the direct correlation of CSDs with protein conformation. Only with an appropriately low charge could proteins adopt a well-folded secondary structure, and protein ions with a much higher charge would be more prone to the loss of secondary structure. Therefore, we believe that the protein CSD changes from offline and online heating indicated their gradual unfolding, although it was not significantly different than LHE (data shown below). 8

9 Figure S5. Generation of LHE via inductive and capacitive coupling. a) The equivalent circuits for the LHE setup, consisting of one inductor (L) and one capacitor (C). The inductor is a ring electrode with an input of high pulsed potential (U 0 ) to provide the LHE power (U 1 ) via electromagnetic induction. The capacitor is composed of the sample solution-air-ms inlet. The resistance of the sample solution is equivalently illustrated by R 2. GND, ground. b) Low-frequency (f 3 ) potential facilitates the generation of LHE. The effective LHE duration (t 1, t 2, t 3 ) is indicated by the frequency-dependent shadow. Due to f 1 > f 2 > f 3, the LHE duration ranks as t 1 < t 2 < t 3. U onset is the threshold potential for nanospray. U 0-p is the amplitude of the pulsed potential (U 0 ). Circuit design for LHE. To further understand the generation of LHE in our strategy, we also drew the equivalent circuit (Figure S5a) of our LHE setup. Previously, a capacitive coupling model with two capacitors was proposed to reveal the underlying principle of contactless spray ionization. 16 Inspired by that model, we proposed that the generation of LHE is mainly originated from the thermal effect of charging processes via inductive and capacitive coupling. The inductive coupling effect enables the transition of pulsed potential (U 0 ) into the driving force (U 1 ) of LHE. The input of pulsed potential on the ring electrode (L) generates an alternating current in the ring-electrode inductor. Thus, an alternating magnetic field is produced near the inductor, and this magnetic field further induces the generation of a strong electric field (U 1 ). Along with the inductive coupling, the capacitor (C) is under electrostatic charging before U 1 reaches the threshold (U onset in Figure S5b) for nanospray, and it is believed that the rapid charging processes make a significant contribution to the LHE of protein samples. Thus, before the nanospray is initiated, the sample solution (R 2 ) is under LHE derived from the thermal effect via the rapid charging process. Once nanospray has occurred (the potential reaches the threshold), LHE stops because the 9

10 charging process has stopped, and the entire process will be continually repeated for the following cycles. Because LHE occurs before the potential reaches the threshold (U onset ), the LHE efficiency depends on the duration (t 1, t 2, t 3 ), and low-frequency (higher duration) potential will thus generate more LHE than high-frequency potential. This process is illustrated in Figure S5b. Furthermore, the amplitude of the pulsed potential significantly affects the LHE efficiency. That is, high-amplitude potential drives more charge accumulation than low-amplitude potential. Thus, high-amplitude potential facilitates the generation of LHE. The effects of potential (U 1 ) frequency and amplitude on LHE are somewhat similar to a previous report claiming that a potential with a relatively low frequency and high amplitude produces a more intense electric field. 17 Additionally, this thermal effect is indeed beyond the measurable range because the charge is even less than 1.0 nc for one pulse (100 Hz input, 10 milliseconds with less than a 0.1 µa current). In brief, the generation of LHE in our strategy is probably the result of the concomitant inductive and capacitive coupling among the ring electrode, sample solution and grounded MS inlet. 10

11 Figure S6. Representative mass spectra derived from the frequency-dependent analysis of NH 4 HCO 3 (2.5 mm)-buffered cytochrome c. 11

12 Figure S7. Voltage-dependent analysis (1000 Hz) of cytochrome c (buffered with 150 mm NH 4 HCO 3 ) with LHE-driven Sequential unfolding. 12

13 Figure S8. Frequency-dependent analysis (3.0 kv 0-p ) of cytochrome c (buffered with 150 mm NH 4 HCO 3 ) with LHE-driven Sequential unfolding. 13

14 Figure S9. Frequency-dependent analysis (3.0 kv 0-p ) of cytochrome c (buffered with 150 mm NH 4 OAc) with LHE-driven sequential unfolding. 14

15 Figure S10. IMMS evidence for Sequential refolding of acid-unfolded Mb. a) Mass spectrum for formic acid-unfolded Mb and ammonium acetate-refolded Mb. The mass spectrum for native Mb in 10 mm NH 4 OAc (AAT, marked Native Mb ) is also shown at the top of a). b) The combined CCSs as a function of charge state. c-e) Respective CCS-charge state curves for the four refolding conditions in a). AAT-induced refolding of Mb (pre-denatured with 0.5% FA) shows a similar CCS transition curve to that of LHE-driven Sequential unfolding, as shown in Figure 3. The theoretical CCS values were obtained from IMPACT calculation with PDB ID of 1MBN. All mass spectra were derived from normal nesi (dc 1.5 kv). We performed a series of refolding experiments for acid-treated Mb to deduce the unfolding processes 15

16 of holo-mb in solution. Shown in Figure S10a are representative mass spectra of NH 4 OAc-induced refolding of acid-unfolded Mb. With 0.5% (v/v) FA, Mb was completely denatured and adopted broad CSD as high as 25+ in the apo-form, in which the heme group is completely detached via acid disruption in solution. Interestingly, when we added NH 4 OAc (10 mm, shown in Figure S10a) to the denatured Mb solution, the apo-mb was observed with gradual refolding indicated by both the CSD shift and the corresponding IMMS measurements (Figure S10b). Surprisingly, when 100 mm NH 4 OAc was added to the denatured Mb solution, almost all the denatured Mb was transferred from apo-mb to native holo-mb. This unique Mb form, refolded by NH 4 OAc, resembled the native Mb buffered with 10 mm NH 4 OAc (bottom of Figure S10a) in both the CSD and CCS values of the corresponding charge states (Figure S10b). 16

17 Figure S11. Mass spectra of myoglobin (Mb) in 1 mm NH 4 OAc derived from normal nesi-ms without LHE. All results from various dc voltages reflect natively folded holo-mb. Figure S12. Sequential unfolding of native-like human insulin. By increasing the frequency (from 3500 Hz to 1000 Hz) of LHE, the unimodal CSD was gradually right-shifted to higher charge states, indicating its gradual unfolding. 17

18 Figure S13. Sequential unfolding of Aβ (1-40) (aqueous solution). Sequential unfolding of human insulin and Aβ (1-40). By gradually decreasing the frequency of LHE, a series of CSD profiles was observed (Figure S12). The most abundant charge increases were from 4+ to 6+, indicating the Sequential unfolding of insulin in solution. It should be noted that the slight variations in the insulin CSD profile may be related to the slight changes in conformation, which in turn validates the stabilizing effect of three disulfide bonds. 4, 18 A similar slight unfolding of insulin was also recently observed using hydrogen-deuterium exchange. 19 Furthermore, the sequential unfolding of Aβ (1-40), which is one of the two main alloforms of the amyloid β peptide widely implicated in Alzheimer s disease, was achieved using LHE in solution (Figure S13). 18

19 Figure S14. Sequential unfolding and ligand detachment of holo-mb in 0.1% DMSO-containing aqueous solution. Voltage-dependent (a, 1 khz) and frequency-dependent (b, 1 kv 0-p ) Sequential unfolding of Mb was processed with the control without LHE (nesi, dc 1.5 kv). Sequential unfolding of holo-mb and resultant detachment of heme group. As mentioned above, one result of the Sequential unfolding of Mb was a gradual change in ligand-binding ability. Therefore, we tentatively studied the binding behavior of Mb upon Sequential unfolding driven by LHE. To achieve this goal, we subjected holo-mb to LHE-driven unfolding in a 0.1% DMSO aqueous solution. We selected DMSO for the following two reasons: 1) DMSO is a commonly used additive in protein assays and drug discovery; and 2) the presence of low concentrations (< 2%) of DMSO increases protein charges, and higher concentrations (> 2%) lead to supercharging Herein, we added 0.1% DMSO to the native-like Mb system, followed by a series of LHE. At low amplitude (1 kv 0-p ) (Figure S14a), compact structure was indicated by narrow CSD centered at 9+, but with a small variation from that of NH 4 OAc-buffered Mb (centered at 8+) (Figure S11). The onset of native-like holo-mb unfolding (Figure S14a) occurred at a voltage of 1.5 kv 0-p, in which a significantly broader holo-mb CSD and the appearance of apo-mb were observed in the mass spectrum. To increase the amplitude of LHE voltage (Figure S14a), the profile of holo-mb CSD was gradually changed from unimodal to trimodal and bimodal, eventually ending with a unimodal type of extensively unfolded state, which exhibited both holo-mb and apo-mb with a broad CSD centered at 19+ (4 kv 0-p ). These observations indicated that the gradual detachment of the heme group occurred concomitantly with the Sequential unfolding of holo-mb. Furthermore, frequency-dependent experiments were performed for the DMSO-containing Mb, the same solution used in the abovementioned voltage-dependent mode. Figure S14b shows that, by decreasing the frequency of LHE voltage, the charge state of holo-mb gradually increased along with a significant increase in the relative intensity of apo-mb, indicating the gradual unfolding of holo-mb along with its dissociation into apo-mb. 19

20 Figure S15. Native nesi mass spectra of 10 μm CaM (in 100 mm NH 4 OAc) after the addition of CaCl 2 at various concentrations. Each column from right to left shows deconvoluted mass distributions for high-charge states (14+ and 13+) and low charge states (7+ and 6+). The number of calcium ions corresponding to the individual peaks is denoted as 4, 3, 2, 1, and 0 (apo). The peaks marked with an asterisk represent CaM ions with sodium adducts. All MS spectra were obtained from dc nesi (2 kv). LHE-driven Sequential unfolding of CaM in solution The LHE-induced thermal unfolding, however, not only works for single-domain proteins and their ligand-binding protein complexes, but also can be extended to monomeric proteins with more than one domain. As a proof-of-concept, CaM is selected as a model protein with four EF-hand motifs, each of which binds one Ca CaM has been known as a multifunctional intermediate messenger that transduces signals by binding calcium. Here, we subjected CaM to LHE-induced thermal unfolding and then tracked its Sequential unfolding processes. Figure 4a shows the unfolding curves of CaM under various buffer conditions. When water was used, CaM adopts almost random secondary structure and thus showed a LHE-independent trend in the Sequential unfolding curve. However, when buffered in concentrated electrolyte solutions (e.g. 100 mm ammonium acetate doped with various contents of NH 4 HCO 3 ), a well-structured CaM was probed and distinct LHE-dependent Sequential unfolding curves can be plotted (Figure 4a). Interestingly, the 20

21 unfolding extent not only highly depends on LHE voltage, but also shows high correlation to the concentration of NH 4 HCO 3 in the buffered solution. For example, at a certain LHE voltage of 2.0 kv 0-p, the unfolding ratios of CaM are 1.2%, 34.3%, 54.2% and 96.8% for the buffers of 100 mm NH 4 OAc, 100 mm NH 4 OAc doped with 10 mm NH 4 HCO 3, 100 mm NH 4 OAc doped with 100 mm NH 4 HCO 3 and water, respectively. This observation indicates ammonium bicarbonate might help protein unfolding in solution, which was also observed in electrothermal unfolding experiments. 27 Those facts further strengthened the effectiveness of thermal unfolding initiated by LHE. In order to correlate LHE-induced CSD variations to solution-phase structural changes of CaM, we then calculated the binding stoichiometry (Figure 4b) and the sequential binding constants (Figure 4c) as a function of LHE voltage and charge state. As shown in Figure 4b, the average binding number of Ca 2+ is plotted to LHE voltage for charge states varying from 6+ to 14+. From Figure 4b, it was observed that the average binding number of CaM with a certain charge is not highly sensitive to LHE voltage. For example, CaM with 7+ charge binds 3.28 Ca 2+ on average at 1.5 kv 0-p LHE, and still binds 3.08 Ca 2+ on average at 3.0 kv 0-p LHE. There is no significant change in Ca binding number of CaM with certain charge numbers during LHE operations, which show similar Ca binding numbers with that under no LHE operations (dc nesi data in Figure 4b). In another word, the LHE operation does not change the nature of CaM ions, such as its ligand-binding behaviors and thus structures of CaM with certain charge numbers. Therefore, the LHE-induced CSD variation is not simply an artifact resulting from charge/proton reactions. Instead, LHE generates thermal unfolding of CaM and thus induces the gradual conformational change in solution. Notably, a distinct relationship between the binding stoichiometry and charge state can be readily obtained from Figure 4b. For the CaM with charges of 6+ and 7+, the average binding number is about Meanwhile, the average binding number is less than 2.5 for CaM with charges from 8+ to 14+. That is, higher charged CaM adopts less compact structures and was observed with less average binding number of Ca 2+, and vice versa. This phenomenon confirmed the tight relationship between CSD and solution-phase structures of CaM, and further indicated LHE-induced CSD variations are highly protein structure-correlated. To further validate the CSD-dependent CaM structure, in addition to binding stoichiometry, the sequential binding constants have also been determined as a function of charge state by using a series of titration experiments (Figure S15). As shown in Figure 4c, the measured overall binding constants are 0.041±0.001, 0.050±0.001, 0.018±0.002 and 0.015±0.002 µm -1 for K1, K2, K3, and K4, respectively. Those overall constants are largely in line with previous report, which reported 0.10, 0.07, 0.03 and 0.08 µm -1 for K1, K2, K3, and K4, respectively. 26 The slight decrease of K values measured in this study is believed to be the result of the detachment of nonspecific CaM-Ca binding artifacts by using a statistical method (detailed deconvolution process can be found in Supplementary Information). Interestingly, we 21

22 found a distinct CSD-dependent K values among all CaM charge states studied here, especially observed with significant differences between less charged CaM (6+/7+) and highly charged CaM (e.g. 10+~14+). For CaM with charges of 6+ and 7+, the specific binding constants of Ca 2+ are highly structure correlated. For example, the K2 and K4 values are far higher than K1 and K3, which indicates the cooperative and sequential binding of Ca 2+ into the two EF hands within CaM. Notably, it is striking that K2 value is larger than K4 derived from both CaM 6+ and CaM 7+, which strongly suggested the unique structural feature that the C-terminal lobe binds Ca 2+ with a three- to fivefold higher affinity than the N-terminal lobe. 22 However, almost no useful structural information could be obtained from the K values derived from CaM with higher charges (e.g. 10+~14+). Those binding constants derived from highly charged CaM seemingly indicate a random incorporation of Ca 2+. The CSD-dependent binding constants (Figure 4c) of CaM are in well accordance with the binding stoichiometry (Figure 4b) in the aspect of correlation to solution-phase CaM structure, but binding constant data have given more detailed binding information. 22

23 Figure S16. Refolding and unfolding of CaM in 100 mm NH4OAc solution. The data are representative mass spectrum from average of 1.5 min data acquisition. Refolding of CaM can be achieved through regulating the amplitude and frequency of LHE voltage. Figure S17. Refolding of Mb containing 0.1% DMSO in 1 mm NH4OAc solution. Both charge state distribution and corresponding collisional cross section indicate the refolding of Mb from regulating the LHE amplitude. Frequency, 200 Hz. 23

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