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Probing Activated and Non-Activated Single Calmodulin Molecule under a picoNewton Compressive Force Susovan Roy Chowdhury, and H. Peter Lu Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01283 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018
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Biochemistry
Probing Activated and Non-Activated Single Calmodulin Molecule under a picoNewton Compressive Force S. Roy Chowdhury, H. Peter Lu* Bowling Green State University, Department of Chemistry, Center for Photochemical Sciences, Bowling Green, OH 43403 *Corresponding Author: E-mail:
[email protected] Supporting Information ABSTRACT: Interrogating protein structure and function inter-relationship under a picoNewton force manipulation has been highly promising and informative. Although protein conformational changes under pulling force manipulations have been extensively studied, protein conformational changes under a compressive force have not been explored in detail. Using our home-modified sensitive and high signal-to-noise AFM microscopy approach, we have applied a picoNewton compressive force, manipulating a CaM molecule to characterize two different forms of 2+ Calmodulin, the Ca -ligated activated form, and the 2+ Ca free non-activated form (Apo-Calmodulin). We observed sudden and spontaneous structural rupture of Apo-CaM under compressive force applied by an AFM tip, though no such events were recorded in case of the 2+ Ca -ligated activated CaM form. The sudden spontaneous structural rupture under a picoNewton force compression has never been reported before, which presents an unexplored function that is likely important for protein-protein interactions and cell signaling functions.
Beyond the static protein structure function relationship, protein conformational dynamics also play very crucial roles 1-7 in biological functions. Single molecule force spectroscopy has advanced into a powerful method for investigating 8-14 protein structure-function relationships. Mechanical force on a single protein molecule originated from the molecular partition crowding, cell osmotic pressure, and cell entropic 15-18 surface tension can be critical in living cells. Generally, mechanical force applied by AFM can be both compressive and pulling force. Both force manipulation experiments have provided important informations about the mechanical, chemical, and structural properties of protein molecules. While, single protein molecules under pulling force manipulation have been studied extensively, there are only a limited number of studies on the consequences of compressive force applied by an AFM tip on a single protein molecule. Here we report structural change of a single 2+ 19 Calmodulin molecule in presence and absence of Ca ions probed by our compressive force curve measurements using AFM.
Calmodulin is a ubiquitous calcium binding protein with 2+ 148 residues (16.7 KDa) which plays crucial roles in its Ca 2+ 20, 21 ligated activated form in transduction of Ca signals. It performs this role by binding to several targets inside the cell including ion channels and a large number of enzymes and proteins. 2+
The crystal structure of Ca -ligated CaM has a very distinct dumbbell shape, where two approximately symmetrical globular C- and N- terminal domains are 22 separated by a 27 residue long α-helical linker (Figure 1A). Both globular domains contain two EF-hand motifs, and 2+ each of these motifs binds with one Ca ion to sense intracellular calcium level. This elongated dumbbell 2+ conformation of Ca -ligated Calmodulin exposes two hydrophobic patches centered on the concave surface of each 23, 24 lobes, which help the molecule to bind with ligands and 25-27 activate a range of kinases.
2+
Figure 1. (A) Crystal structure of Ca -ligated Calmodulin. (B) NMR structure of Apo-Calmodulin in solution. 2+
When Ca ions are removed from the EF hand motifs of Calmodulin, it transforms to a more bound conformational state Apo-CaM from its prominent dumbbell shape (Figure 1B). It is clear from NMR study that both lobes of Apo-CaM form a globular four helix-bundle conformation. In this 28, 29 closed conformation hydrophobic residues are inaccessible to external ligands.
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In our experiment of compressive force manipulation on 2+ both Apo- and Ca -ligated CaM, we used a home-modified AFM apparatus with an ultra-soft AFM tip to apply compressive force on a single protein molecule tethered to a glass coverslip through covalent bonding between the linker molecule and amino group of the protein (Figure S1) and studied its response. We found that Apo-CaM molecules undergo through an abrupt spontaneous rupture at ~70 pN (Supporting Information, Figure S2) of compressive force. Figure 2 shows a general pattern of force curve corresponding to a single Apo-CaM protein rupture and a cartoon scheme of AFM tip-protein interaction in the process of force loading on a protein molecule. It was also observed 2+ that force loading on a single Ca -ligated CaM molecule does not go through any such rupture event.
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the rigidity and enable the molecule to withstand pN amount of force. Significantly, we observed an abrupt force drop when the AFM force loading on the single Apo-CaM molecule reaches a certain threshold value. This abrupt drop in the force curve corresponds to the sudden release of force on the AFM tip, i.e. the protein molecule can no longer hold the force at the threshold value, which causes a simultaneous and spontaneous collapse of significant amount of intramolecular interactions and hydrogen bonds which hold the molecule together. This type of protein rupture events under compressive force have never been reported previously. Nevertheless, this protein property is highly significant and closely related to protein functions in living cells, as the thermal fluctuations of local force may provide such pN force fluctuations and trigger protein structure collapse, a catastrophic unfolding event, that may be associated to protein dysfunction, aggregation and misfolding. 2+
We carried out the same experiment on Ca -ligated CaM. Figure 3B represents the typical AFM compressive force 2+ curve on a Ca -ligated single CaM molecule. There is no abrupt force drop in this force curve, which signifies the absence of rupture event under compressive force. We also noticed that after treatment with EGTA, which takes away 2+ 30 Ca ions from Calmodulin converting it to Apo-CaM, the rupture event was recovered (Supporting Information, Figure S3). Nevertheless, we have also noticed repeated rupture events on a single protein molecule when we carried out a repeated force manipulation experiment on the same protein molecule. Which suggests that the ruptured protein likely refolds back to its native state after the force is removed. Figure 2. (A) The mechanical force curve of AFM tip interaction with a single Apo-CaM molecule. A rupture event was recorded in this curve. (iii) The AFM tip approaches to the single Apo-CaM molecule. At the point ‘ii’, the mechanical force loading starts as the AFM tip touches the surface of the targeted Apo-CaM molecule. (iiiii) The force loading continues to a certain threshold value. At point ‘iii’ the loading force on the protein reaches the threshold value and the protein cannot hold the force anymore and gets spontaneously ruptured, resulting the loaded mechanical force abruptly drops to ‘iv’; (ivv) The force loading resumes when the AFM tip touches the coverslip glass surface; (vivii) This part of the force curve represents typical AFM tip pulling up from the sample surface. (B) Cartoon scheme of the interaction of AFM tip apex and the Apo-CaM molecule. ‘ii’ represents the point where the compressive force loading starts on the protein. ‘iii’ represents the point where the force loading reaches a threshold value and the protein structure spontaneously ruptures. ‘iv’ represents the point where the loading force abruptly drops due to protein rupture. Figure 3A shows a typical AFM force curve where an AFM tip approaches a single Apo-CaM molecule. Our AFM apparatus has sufficient sensitivity and signal-to-noise ratio to record the entire pN level of force loading on a single protein molecule. Intramolecular hydrogen bonding, interdomain friction force, liquid friction force with the solvent molecules and intermolecular hydrogen bonding between protein and the solvent molecules contributes to
Figure 3. AFM force curve of compressive force manipulation on single protein molecule. (A) AFM force curve of a single Apo-CaM molecule which shows a rupture event under compressive force. (B) AFM force curve of a 2+ single Ca -ligated CaM molecule, which did not show any rupture event under compressive force manipulation. 2+
Although the crystal structure of Ca -ligated Calmodulin shows the central helix in α-helix form, but NMR structure conclusively showed this linker is nonhelical and very flexible around its middle point, from residue K77 to S81. The
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Biochemistry anisotropy observed for the motion of the two lobes was 2+ much smaller, which indicates that in Ca -ligated Calmodulin, tumbling of both N terminal and C terminal 31 lobes are mutually independent. This flexible central helix structure model was further supported by structure of CaM molecule complexed with target peptides, where these target peptides induce collapse of the elongated dumbbell structure forming a globular structure around the helical target 32-34 2+ peptide. In presence of Ca ions, this extended flexibility gives the CaM protein the conformational freedom where it can release the tension and avoid an abrupt rupture under 2+ compressive force. In other words, in the presence of Ca , a CaM molecule behaves more like a nonrigid sphere where it can easily change its shape redistributing the loading force applied by the AFM tip. As a result, the molecule does not go through a structural rupture under a compressive force. 2+
On the other hand, in absence of Ca , the central helix of Apo- CaM is significantly less flexible which forbids both the domains to come together and bind to the peptide. The significantly larger degree of anisotropy in rotational 2+ diffusion observed for Apo-Calmodulin relative to Ca ligated Calmodulin further concludes that the linker is more 2+ 35 rigid in the Apo state compared to the Ca activated state. The Apo-CaM is in a more bounded state, which contributes 2+ to its structural rigidity whereas Ca -ligated Calmodulin is in an open state. When the force reaches to a threshold value of ~70 pN, the CaM can no longer hold the force, and the molecule gets ruptured spontaneously and abruptly. Protein rupture under compressive force is a spontaneous process driven by a threshold amount of force. As the AFM force loading is very slow 1.5 nm/ms, temperature around the protein remains constant and the rupture dynamics follow a typical energy profile with an energy crossing barrier which includes complex nature of dynamic bond breaking, intramolecular interaction dynamics, and liquid friction force. The inhomogeneous nature of the protein rupture indicates the inhomogeneous local environment constituted by different orientations of the protein molecules along with different electric, hydrophilic and hydrophobic force fields of the single protein molecules, solvent molecules and the linker molecules on the cover glass surface. Structural rigidity attributed from interdomain interactions, hydrogen bonds, and solvent dynamics is very important to study protein structure-function relationship associated with protein-protein, protein-peptide interactions and enzymatic reactions. Figure 4A shows the Gaussian distribution of the rupture force of single Apo-CaM molecules under 1500 nm/s approaching velocity. From this distribution, we calculated the rupture force which was around 70 pN. We note that this rupture force is dependent on the AFM tip approaching velocity. To calculate the actual threshold force loading distance, i.e. the amount of structural change of the targeted Apo-CaM molecule under the process of compressive force loading to the threshold value, we analyzed the force curves considering two factors. Firstly, the force loading process causes the tip bending. Because of that, distance traveled by the electropiezo scanner exceeds the distance traveled by the AFM tip apex. The amount of tip bending in process of force loading can be easily taken in account considering the force constant of the cantilever (30 pN/nm). Secondly, the
contribution of the hydration shell around a protein 36, 37 Therefore, in our recorded molecule (1-2 nm of width). force curves, force increases in a much slower rate at the beginning which corresponds to the AFM tip interaction with the outer solvation layer of the protein. In the latter part of the force curve, the force increases in a much faster manner as the AFM tip starts to interact with the protein surface directly. Although identifying the atomic level contact of the AFM tip with the protein surface from our force curve measurements was beyond the scope, we assume that the contact occurs when the compressive force loaded by the AFM tip reaches a certain value to counter the water solvation layer. We plotted different assessments in Figure 4B from a chosen “force upon contact” at 0, 5, 10, 15, 20, and 25pN. Figure 4C shows the distribution of electropiezo displacement, which was read out directly from the force curve. It is the distance between the point where force loading starts and the point where protein rupture occurs. However actual distance of force loading to the threshold value is significantly shorter because of the electropiezo displacement calibration. We calculated the loading energy using ܧௗ ൌ ܨ ሺ݈ሻ݈݀. Where l is the compressive force loading distance, and ܨሺ݈ሻ is the loading force. Notably, the force loading distance is essentially at least few nm less than the AFM electropiezo displacement (Figure 4C) due to tip bending. Although the actual nature of the force loading curve on a single Apo-CaM is nonlinear, in our calculation, we assumed that force increases linearly during the loading process in our data analysis. Under this approximation, distribution of the calculated threshold compressive force loading energy was plotted in figure 4D using Eloading= (F threshold/2)×lthreshold and the mean value was around 32 kBT; however, the energy can be as low as 10 kBT with significant probability, which is biologically accessible from the local thermal fluctuations, such as in living mammalian cells at o 37 C.
Figure 4. The characterization of compressive force loading. (A) Distribution of threshold force of Apo-CaM rupture under 1500 nm/s AFM tip approaching velocity. (B) Estimated average force loading distance after adjustment with standard deviation, under the assumption that the atomic level contact between AFM tip and the Apo-CaM molecule occurs after the force reaches 5,10,15,20, and 25pN, respectively to counter the hydration shell (Figure S4). Negative sign represents the AFM tip position before contact with the protein solvation layer. Average threshold force
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loading distance was 3.8 nm. (C) AFM electropiezo displacement distribution, which is defined as the distance traveled by AFM electropiezo from the start of the force loading on a targeted Apo-CaM molecule to the point of rupture. Though, actual distance of force loading is significantly shorter due to the electropiezo displacement calibration. (D) Distribution of calculated loading energy under 1500 nm/s loading velocity. All histograms (A, C & D) are fitted with Gaussian function. Structural flexibility and rigidity of Calmodulin molecule play a very important role in protein function associated with binding other proteins and peptides. There are numerous attempts have been made over the years to address the 2+ flexibility of Calmodulin in presence and absence of Ca using NMR and other techniques, but it is still hotly debated due to the absence of direct evidence. It is significant that the structural rigidity of a protein molecule can be probed by using a compressive force. It further proves that the compressive force is equally sensitive like pulling force to sense such miniscule amount of structural change in terms of flexibility and compactness. Furthermore, we also observed 2+ spontaneous single protein rupture of bound Ca deactivated form under compressive force which could be a plausible mechanism leading to protein misfolding and entangled aggregation. In summary, we have utilized AFM compressive force to manipulate and characterize both calcium activated and deactivated forms of Calmodulin. Upon loading of compressive force on a single Apo-CaM molecule we observed an abrupt and spontaneous rupture of the protein, which is an unexplored property of the protein. On the other 2+ hand, we observed no such events in case of Ca -ligated 2+ form, i.e. Ca -ligated form is more flexible which makes it unable to hold force. This protein property is highly significant in protein functions in living cells, as the thermal fluctuation local force fluctuations may provide such pN force and trigger a protein structure collapse or a multiple protein collapse simultaneously at the same location, a catastrophic unfolding event, that may be associated to 15, 18 protein dysfunction, aggregation and misfolding. ASSOCIATED CONTENT Supporting Information Additional information, the experimental section, and figures are included in supporting information. (PDF) AUTHOR INFORMATION
Corresponding Author *Bowling Green State University, Department of Chemistry, Center for Photochemical Sciences, Bowling Green, OH 43403 E-mail:
[email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work is supported by the Ohio Eminent Scholar Endowment. REFERENCES
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