Reliable Tracking In-Solution Protein Unfolding via Ultrafast Thermal

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Reliable Tracking In-solution Protein Unfolding via Ultrafast Thermal Unfolding/Ion Mobility-Mass Spectrometry Gongyu Li, Shihui Zheng, Yuting Chen, Zhuanghao Hou, and Guangming Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00859 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Analytical Chemistry

Reliable Tracking In-solution Protein Unfolding via Ultrafast Thermal Unfolding/Ion Mobility-Mass Spectrometry Gongyu Li,†,§ Shihui Zheng,†,§ Yuting Chen,† Zhuanghao Hou,† and Guangming Huang*,†,‡ † Department of Chemistry, School of Chemistry and Materials Science and ‡National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China

Supporting Information Placeholder ABSTRACT: Sequential unfolding of monomeric proteins is important for the global understanding of local conformational elements (e.g., secondary structures and domain connections) within those protein assemblies. Ion mobility-mass spectrometry (IM-MS) is an emerging and promising technique for probing gradual protein structural perturbations in the gas phase. However, it is still challenging to track sequential unfolding in the solution phase. Here, we extended IM-MS to track in-solution sequential unfolding of monomeric proteins having single and/or multi-domains. The present method combines ultrafast local heating effect (LHE)-driven sequential unfolding with IM-MS identification. Protein sequential unfolding in solution is demonstrated by the rapid and controllable IM-MS data switch between native and gradually unfolded states. Our results show that LHE induces gradual protein conformational transitions associated with biological functions, where IM-MS tracks the sequential unfolding of monomeric proteins.

In the solution phase, proteins with well-defined native structures are often stabilized by a set of weak intramolecular and intermolecular interactions. Although most static protein structures have been well resolved using established spectroscopic techniques (e.g., X-ray crystallography and cryo-electron microscopy),1-2 it is always more desirable to accurately understand dynamic protein structures by precisely manipulating their spatial arrangements. Monomeric proteins are commonly used to study protein folding and unfolding since they simplify the folding processes (conformational transitions) and thus allow potential in-depth investigation without complications from adjacent proteins. The major obstacles for the sequential unfolding of monomeric proteins include i) how to rapidly and controllably trigger monomeric protein unfolding in the solution phase and ii) how to accurately identify those conformational transitions (which requires delicate delivery of target proteins from the solution phase to the structural detection zone). Recently, significant efforts have been successfully devoted to gas-phase conformation manipulation using collision-induced unfolding-ion mobility-mass spectrometry (CIU-IM-MS).3-5 However, in most cases, the sequential unfolding of monomeric proteins occurs after the formation of gas-phase ions. Although gas-phase unfolding can be correlated with the solution-phase structures, direct sequential unfolding in the solution phase has not yet been achieved. Effective means of sequential unfolding monomeric proteins in the solution phase include the introduction of chaotropes (surfactant, urea, bases and acids) and thermal unfolding.6-10 While the addition of chemical reagents in solution tends to both unfold and denature proteins,11 thermal unfolding produced more detailed transient intermediate information about “native-like” proteins in the solution phase when the heating process was reduced to ~10 minutes.12 Although significant success has been achieved using thermal unfolding/IM-MS measurements, much wider impacts other than fundamental studies could be achieved if we could, for example, link the functions (such as binding behaviors with ligands and metal ions) and structures of proteins

during sequential unfolding. Thus, we aim to develop a rapid (millisecond level) thermal unfolding/IM-MS method for the sequential unfolding of monomeric proteins that is sufficiently rapid to maintain bound ligands or metal ions.

Figure 1. Ultrafast thermal unfolding/IM-MS-based strategy for tracking the sequential unfolding of monomeric proteins in solution. a) The modified nESI-IM-MS working platform. LHE of protein solutions occurs in a nanospray emitter. Then, the sequential-unfolded proteins are transferred to the gas phase within a millisecond time scale for IM-MS identification. b) Illustration of in-solution sequential unfolding of monomeric proteins with LHE and/or chemical unfolding. Inspired by previously reported thermal unfolding designs,12-16 we modified our home-built circuit17-21 to generate local heating effect (LHE) prior to IM-MS measurements, which provides a millisecond-level thermal unfolding-based method to initiate the sequential unfolding of monomeric proteins in solution. To the best of our knowledge, we are the first to simultaneously achieve

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both i) in-solution sequential unfolding (via ultrafast heating) and ii) gas-phase structural monitoring (via IM-MS-based CCS measurements) for native-like proteins. Previous studies have focused on either “slow” heating-induced unfolding (in solution phase) followed by IM-MS measurements,12 or gas-phase “fast” unfolding in conjunction with IM-MS measurements, like collisioninduced unfolding (CIU). Thus, the major differences between present technique and previous studies are the location and speed of heating. The in-solution characteristic would be beneficial for revealing “real” protein unfolding events, and the extremely narrowed heating time window would help capture more sequential protein unfolding intermediates. In addition, due to the features of non-contact between proteins and electrode, rapid heating and controllable switch, the LHE originating from electromagnetic induction does not induce significant oxidation of native proteins,22 while native proteins might undergo unexpected oxidation during other external heating processes.14, 23-24 Therefore, our strategy provides an opportunity to rapidly study the sequential unfolding of “native-like” monomeric proteins in solution and without oxidation interference. This newly developed ultrafast thermal unfolding method was assembled into a normal nanoelectrospray ionization (nESI) source, which then enables rapidly switchable native and gradually unfolded (prior to denaturing) MS data to be obtained from a single protein solution. In a word, the proposed method (shown in Figure 1) involves i) rapid sequential unfolding of monomeric proteins in solution and ii) reliable structural identification using IM-MS. The workflow for tracking the sequential unfolding of monomeric proteins in solution is illustrated in Figure 1a. A snapshot of our setup can be found in Figure S1. Cytochrome c (cyto c, a single-domain protein) was chosen as the first model monomeric protein since its unfolding intermediates and resulting CSDs are well known with a transition from a tightly folded, narrower CSD (centered approximately 8+) to an unfolded, broader CSD (centered approximately 16+) upon decreasing the pH to as low as 2.6.10 We found that increased protein unfolding was obtained with a relatively low frequency and high amplitude, which was believed to produce a more intense electric field, as reported by previous studies.25 The gradually changed structure could be observed as its CSD changed from a unimodal to a bimodal profile for voltage-dependent analysis (Figure S2a, c). LHE could tune the sequential unfolding upon applying voltages within hundreds of milliseconds (Figure S2a). Its rapid tuning could be quite difficult for the conventional chemical unfolding techniques (Figure S2b, d). Thus, the rapid and controllable feature allows us to study protein conformations in a manner of sequential unfolding before partially denaturing the protein in bulk solution. Recently, similar electrothermal unfolding of proteins during droplet transfer at the MS inlet was reported,26 while LHE in our strategy can achieve rapid thermal unfolding in bulk solution.

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Figure 2. Sequential unfolding of monomeric cyto c in solution tracked via ultrafast thermal unfolding/IM-MS as indicated by CSD shift (a) and CCS variation (b). a) Chronographs of folded (5+~7+) and unfolded (8+~18+) cyto c and their ratios during sequential unfolding via frequency-dependent manipulation (from 4 kHz to 0.1 kHz, 2 kV0-p). Scale bar, 6 seconds. The corresponding mass spectrum is shown in Figure S6. b) The CCS-charge state curve of sequential-unfolded cyto c. Only dominated charge states’ CCS values were plotted. The experimental CCS values were calculated using the same method as described in previous publication.27 The theoretical CCS values from helium were obtained from IMPACT calculation with PDB ID of 1HRC. Buffer, 2.5 mM NH4HCO3 aqueous solution. The above-mentioned results suggested that ultrafast heating may contribute to the sequential unfolding of native-like monomeric proteins. The offline and online heating experiments shown in Figure S3 suggested that monomeric proteins would unfold and even release their binding ligands, though the effect was not as significant as with LHE (data shown below). Thus, the offline and online temperature-jump experiments have further verified the basic principle of ultrafast thermal unfolding, though those experiments can only partially the unfolding pathway. The corresponding gas-phase CIU-IM-MS results (Figure S4), namely, charge state-dependent CIU profiles with distinct transitions,4 further validated the change in secondary structures along with the CSD variation derived from heating in solution. Moreover, we tentatively drew the equivalent circuit (Figure S5) of our LHE setup to deduce the generation of LHE in our strategy; a related description can be found in the Supplementary Information. Therefore, we conclude that the thermal effect derived from LHE is one of the most probable mechanisms involved in the sequential unfolding of monomeric proteins in our strategy. In our strategy, there is no need to frequently change protein solutions. Specifically, regulation of only the amplitude and frequency of LHE voltage can produce abundant conformational information from the same protein solution. This result was further verified via a frequency-dependent analysis of the buffered cyto c. Figure 2a shows that the sequential unfolding of monomeric cyto c could be achieved by adjusting the LHE frequency, as indicated by the structurally informative mass spectrum (Figure S6) and corresponding charge state-dependent CCS values (Figure 2b). It should be noted that, upon changing the LHE frequencies, a series of unfolding steps could be accomplished with an estimated equilibrium duration of approximately 6 seconds (the LHE durations for a single spray were less than 300 ms for all of the potentials used here).18 The chronograph in Figure 2a also demonstrated the stability of LHE for the sequential unfolding of monomeric proteins. A similar sequential change in the secondary structure driven by LHE in solution was observed in the CIU experiments for cyto c at charges of 6+ and 7+ (Figure 2c). Proteins with an appropriately low charge are known to have well-folded secondary structures, which could be observed at charges of 6+ and 7+, as shown in Figure S4, and proteins with higher charges (e.g., myoglobin (Mb) at 15+, Figure S4) did not produce reliable information for secondary structures. The charge state-dependent CIU profiles were in good agreement with a previous report.4 Thus, the above-mentioned facts indicate that the sequential unfolding (Figure 2a) driven by LHE in our strategy is closely related to the changes in secondary structures.


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Figure 3. Sequential unfolding of monomeric holo-Mb in solution. To increase the LHE voltage (setting frequency, 1 kHz) from 1.0 kV0-p to 4.0 kV0-p, the sequential unfolding events were captured using nESI-MS (b, d, f) and IM-MS (a, c, e) measurements. The LHE-driven sequential unfolding behavior followed a CCScharge state profile similar to that of chemical unfolding (light yellow region, a, c, e and Figure S10). The experimental CCS values were calculated using the same method as described in previous publication.27 The theoretical CCS values from helium were obtained from an IMPACT calculation with PDB ID of 1MBN. Buffer, 1 mM NH4OAc aqueous solution. Compared with the previous gas-phase unfolding method, this strategy directly depends on the IM-MS readout of CCS values. However, there is no need for the tedious optimization of the charge states of precursor protein ions. Instead, all protein charge states are accounted for, and their CCSs are measured. In addition, our strategy also works for monomeric proteins under relatively high-concentration buffer conditions (150 mM NH4HCO3 Figures S7-8; 150 mM NH4OAc, Figure S9), which favor the preservation of native-like protein conformations with more choices of buffer solutions.28 Then, Mb was selected as our second model monomeric protein because it releases a prosthetic group of heme (MW 615) during its unfolding processes, which results in the transformation of holo-Mb (MW 17565.8) into apo-Mb (MW 16950.8). Previous biophysical studies29 have suggested that the gas-phase stability of the oxygen-carrying protein Mb is similar to its solution-phase state, which is a tightly folded, compact structure. For the first time, we combined a modified nESI with IM-MS to directly observe the CCS values of in-solution unfolded Mb in real time through LHE-based rapid and sensitive method for protein conformation manipulation. Firstly, we conducted LHE-driven sequential unfolding experiments to reveal more detailed conformational changes in holo-Mb. Under low-LHE conditions (1 kV0-p), natively folded holo-Mb (Figure 3a) was observed with a low charge state of narrow CSD (Figure 3b), and the holo-Mb ions had CCS values similar to those expected for native structures (Figure 3a) in solution. By increasing the amplitude of LHE voltage (from 1 kV0-p to 4 kV0-p), the most abundant charge state of holo-Mb was significantly changed from 9+ (Figure 3b) to 10+ (Figure 3d) and finally 13+ (Figure 3f). Then, a gradual confor-

mational change in holo-Mb was indicated by those distinct CSD changes along with the “CCS-charge state” changing trend (Figure 3a, c, e). Notably, this “CCS-charge state” trend is in good agreement with the salt-mediated refolding results shown in Figure S10. The sequential unfolding of holo-Mb driven by LHE was also strengthened by the control experiments (Figure S11): without LHE, no unfolding events occurred. Interestingly, we observed various conformers with different unfolding degrees for a certain charge using IM-MS measurements of LHE-unfolded proteins. For example, at 4 kV0-p (Figure 3e, f), two conformers were observed for both the 9+ and 10+ charge states, which indicated that holo-Mb did unfold when subjected to the LHE treatment. In short, holo-Mb had a native-like conformation in NH4OAc solution, and its native-like structure could be retained under the condition of low or no LHE, while an extended globular structure was probed under higher LHE conditions. In addition to cyto. c and Mb, similar sequential unfolding was tested for human insulin (Figure S12) and Aβ (1-40) (Figure S13). Finally, as demonstrated by the interplay between apo- and holo-Mb (Figure S14), we not only observed the detailed sequential unfolding of holo-Mb but also traced the ligand-binding behavior as a result of the LHE-driven sequential unfolding.

Figure 4. Sequential unfolding of calmodulin (CaM) in solution. a) LHE-induced CaM sequential unfolding under various buffer conditions. NH4OAc, 100 mM. The unfolding ratio was calculated from the intensity of CaM with charges from 8+ to 14+ divided by the total intensity of CaM with all charges. b) Average binding number of Ca2+ as a function of LHE voltage and charge state of holo-CaM (CaM, 10 µM; Ca2+, 40 µM). c) Charge statedependent binding constant of CaM for Ca2+, as measured via titration experiments. d) Sequential binding of Ca2+ into CaM EF hands with four distinct binding constants (K1-K4). The cartoon illustration of the CaM structure was obtained from RCSB with PDB ID of 1CLL and 1CFD. All error bars denote s.d.; n= 3. However, the LHE-induced thermal unfolding not only works for single-domain proteins and their ligand-binding protein complexes but can also be extended to monomeric proteins with more than one domain. As a proof-of-concept, calmodulin (CaM) was selected as a model protein with four EF-hand motifs, each of which binds one Ca2+.30-34 Figure 4a shows the unfolding curves of CaM under various buffer conditions. When water was used, CaM adopted an almost random secondary structure and thus showed an LHE-independent trend in the sequential unfolding curve. However, when buffered in concentrated electrolyte solu-



tions (e.g., 100 mM ammonium acetate doped with various amounts of NH4HCO3), a native-like CaM was probed, and distinct LHE-dependent sequential unfolding curves could be plotted (Figure 4a). Interestingly, the unfolding extent highly depended on the LHE voltage and showed a significant correlation with the concentration of NH4HCO3 in the buffered solution. This observation indicates that ammonium bicarbonate may help track the conformational changes via sequential unfolding in solution, which was also observed in electrothermal unfolding experiments.26 To correlate LHE-induced CSD variations with solution-phase structural changes in CaM, we calculated the binding stoichiometry (Figure 4b) and the sequential binding constants (Figure 4c) as a function of LHE voltage and charge state. Notably, a distinct relationship between binding stoichiometry and charge state could be readily obtained from Figure 4b. Specifically, the more highly charged CaM adopted a less compact structure and exhibited a lower average binding number of Ca2+, and vice versa. This phenomenon confirmed the tight relationship between CSD and solution-phase structures of CaM, and it further indicated that LHEinduced CSD variations are highly protein structure-correlated. To further validate the CSD-dependent CaM structure, the sequential binding constants were also determined as a function of charge state using a series of titration experiments (Figure 4c and Figure S15). Interestingly, we found that, among all the CaM charge states studied here, significantly higher K values (Figure 4c) were observed for less charged CaM (6+/7+). For CaM with charges of 6+ and 7+, the specific binding constants of Ca2+ were highly structure-correlated. For example, the K2 and K4 values were far higher than the corresponding K1 and K3, which indicates the cooperative and sequential binding of Ca2+ into the two EF hands within CaM (indicated in Figure 4d). One striking observation is that the K2 value was larger than that of K4 for both CaM 6+ and CaM 7+, which strongly suggested a unique structural feature in which the C-terminal lobe binds Ca2+ with a three- to five-fold higher affinity than the N-terminal lobe.30 However, the significantly lower K values with CSD over 8+ indicate that CaM with higher charges may suffer from partial unfolding. Hence, it was comprehensively demonstrated that the newly developed strategy, LHE-induced thermal unfolding, also works for CaM, which is a typical model for multi-domain proteins. In summary, as a complementary alternative to chemical and mechanical unfolding, we have presented a versatile ultrafast thermal unfolding/IM-MS strategy for reliably tracking the insolution sequential unfolding of various monomeric proteins under a series of solution conditions. Notably, this technique can also be used for refolding proteins that are pre-unfolded both by using this ultrafast thermal unfolding (Figure S16) and by using solution disruption methods (Figure S17) as indicating by the jump in CSD and CCS/drift time values. The present method may serve as an effective means to generate gradual conformational transitions for “native-like” proteins in the solution phase, which is closely associated with their biological functions. We hope that we have provided a supplementary technique for the pharmaceutical industry and for many biochemical laboratories. We anticipate that this strategy will also be useful for discriminating subtle structural differences among protein analogs and populations; this feature may find an application in ligand binding-based drug screening by combining the present strategy with association constant determinations and binding stoichiometry estimations (e.g., drug-to-antibody ratios). This study reported an alternative for protein conformational interrogation based on ultrafast thermal unfolding and IM-MS measurements. It can be summarized that, the key element in-

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volved in our strategy is the use of electromagnetic induction heating technique, on the basis of our previous relating research experiences.18-19, 35-36 This technique has been assembled into a conventional nanospray platform, which enabled the online structural identification of the ultrafast thermal unfolded proteins. Overall, our strategy has removed the obstacles for tracking insolution sequential unfolding of monomeric proteins. Nanospray, as a widely-used soft ionization technique, serves as the chosen delivery tool to transfer target proteins from solution to structural identification zone. One may concern the potential structural perturbation during nanospray flight processes which involve multistage desolvation steps, though a huge amount of pioneer efforts have devoted to demonstrate the structural preservation of protein structures during millisecond nanospray processes.3, 28, 37-40 In addition, currently, the exact mechanism is not fully understood. We believe that native proteins will undergo a series of heating-triggered unfolding when subjected to AC-induced potential with various amplitudes and frequencies. It would be more persuasive to directly monitor the temperature change of a protein solution along with voltage regulation. However, due to the low power (e.g., heating current less than 0.1 µA) and rapid switching of heating power (e.g., 10-5000 Hz), the overall thermal effect cannot be directly detected at this stage. This initial report does not provide a so-far commercialized method because some aspects leave substantial room for improvement, including temperature monitor, LHE electrode design, power supplier and structural refinement. However, as indicated by the gradual detachment of heme from holo-Mb upon sequential unfolding, the critical link between structure and function of monomeric proteins can be built and this work should have technically provided an experimental foundation.

ASSOCIATED CONTENT Supporting Information Supporting information, including Materials and Methods, figures (Figure S1-S17), offline and online heating experiments, circuit design for LHE and other relating results. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected]

Author Contributions §

G. L. and S. Z. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledged Professor Brandon T. Ruotolo (University of Michigan, Ann Arbor) and Yuwei Tian (University of Michigan, Ann Arbor) for the instrumental support and insightful discussions, and Yang Li (University of Science and Technology of China, USTC) for the kindly assistance of the configuration of equivalent circuit and the relating description of the circuit. We acknowledge National Natural Science Foundation of China (21775143 and 21475121), the Fundamental Research Funds for the Central Universities (WK6030000026 and WK3460000002), the Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology (2017FXCX003), and Recruitment Program of Global Expert and the USTC Graduate School Fellowship for International Exchange (GS006).

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TOC graphic for this manuscript only:

Using Gas-phase Technique to Track In-solution Protein Sequential Unfolding: The present method employs local heating effect to rapidly trigger “native-like” protein sequential unfolding in the solution phase, and the sequential unfolding processes were then captured by online IM-MS measurements.

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Figure 1. Ultrafast thermal unfolding/IM-MS-based strategy for track-ing the sequential unfolding of monomeric proteins in solution. a) The modified nESI-IM-MS working platform. LHE of protein solutions occurs in a nanospray emitter. Then, the sequential-unfolded proteins are transferred to the gas phase within a millisecond time scale for IM-MS identification. b) Illustration of in-solution sequential unfolding of monomeric proteins with LHE and/or chemical unfolding. 99x72mm (300 x 300 DPI)



Figure 2. Sequential unfolding of monomeric cyto c in solution tracked via ultrafast thermal unfolding/IM-MS as indicated by CSD shift (a) and CCS variation (b). a) Chronographs of folded (5+~7+) and un-folded (8+~18+) cyto c and their ratios during sequential unfolding via frequency-dependent manipulation (from 4 kHz to 0.1 kHz, 2 kV0-p). Scale bar, 6 seconds. The corresponding mass spectrum is shown in Figure S6. b) The CCS-charge state curve of sequential-unfolded cyto c. Only dominated charge states’ CCS values were plotted. The experi-mental CCS values were calculated using the same method as described in previous publication.27 The theoretical CCS values from helium were obtained from IMPACT calculation with PDB ID of 1HRC. Buffer, 2.5 mM NH4HCO3 aqueous solution. 99x58mm (300 x 300 DPI)


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Figure 3. Sequential unfolding of monomeric holo-Mb in solution. To increase the LHE voltage (setting frequency, 1 kHz) from 1.0 kV0-p to 4.0 kV0-p, the sequential unfolding events were captured using nESIMS (b, d, f) and IM-MS (a, c, e) measurements. The LHE-driven sequential unfolding behavior followed a CCS-charge state profile similar to that of chemical unfolding (light yellow region, a, c, e and Figure S10). The experimental CCS values were calculated using the same method as described in previous publication.27 The theoretical CCS values from helium were obtained from an IMPACT calculation with PDB ID of 1MBN. Buffer, 1 mM NH4OAc aqueous solution. 99x99mm (300 x 300 DPI)



Figure 4. Sequential unfolding of calmodulin (CaM) in solution. a) LHE-induced CaM sequential unfolding under various buffer condi-tions. NH4OAc, 100 mM. The unfolding ratio was calculated from the intensity of CaM with charges from 8+ to 14+ divided by the total inten-sity of CaM with all charges. b) Average binding number of Ca2+ as a function of LHE voltage and charge state of holo-CaM (CaM, 10 µM; Ca2+, 40 µM). c) Charge state-dependent binding constant of CaM for Ca2+, as measured via titration experiments. d) Sequential binding of Ca2+ into CaM EF hands with four distinct binding constants (K1-K4). The cartoon illustration of the CaM structure was obtained from RCSB with PDB ID of 1CLL and 1CFD. All error bars denote s.d.; n= 3. 99x73mm (300 x 300 DPI)


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TOC 24x6mm (300 x 300 DPI)


Reliable Tracking In-Solution Protein Unfolding via Ultrafast Thermal

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