Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University,

Transcription

1 Supporting Information for Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under PicoNewton Compressive Force Manipulation S. Roy Chowdhury, Jin Cao, Yufan He, H. Peter Lu * Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH *Corresponding Author: hplu@bgsu.edu 1. Structure of HPPK and CaM molecule. Figure S1. (A) Structure of HPPK molecule with FRET dye pair (Collected from Protein Data Bank, and processed with VMD software). Amino acid residues 88 and 142 were mutated with

2 Cysteine and labeled with Cy3 and Cy5 dye respectively. (B) Structure of CaM molecule with FRET dye pair. Amino acid residues 34 and 110 were mutated with Cysteine and labeled with Cy3 and Cy5 dye respectively. (C) AFM image of single HPPK molecules on a cover glass surface ( points/line) and the topography of the encircled single HPPK protein molecule along the yellow line. We prepared the sample with ten times higher HPPK protein population as no optical measurement was involved. Height of the protein molecules on the cover glass was around 5 nm. The width measurement of the protein was affected by image artifacts due to much larger size of the AFM tip. From the height profile radius of the AFM tip curvature was calculated. The radius of the AFM tip curvature was around 33 nm. 2. AFM tip and tip force constant calibration. We used Cr-Au coated AFM Probe (Micromash HQ:CSC38/Cr-Au). There are three cantilevers A, B, C. with force constant 0.09 nn/nm, 0.03 nn/nm and 0.05 nn/nm. We typically used the cantilever B with 0.03nN/nm force constant. We calibrate the cantilever force constant in our experiment by using Thermal K provided by Agilent AFM. Thermal K calculates an AFM probe spring constant by describing the motion of the cantilever as a harmonic oscillator using the equipartition theorem from the theory of fundamental thermodynamics. The curvature of the Cr-Au coated tip has a radius less than 35 nm. 3. AFM Force spectroscopy. S3.1 Single-molecule AFM force spectroscopy analysis

3 Figure S2. The concept of analyzing an experimental AFM force spectroscopy curve to get protein rupture threshold force. As the AFM piezo approaches towards the surface, (a) when the tip is in contact with the protein molecule on the glass surface, a compressive force loading process starts; As the piezo continues to approach toward the surface, the compressive force on the protein molecule increases; simultaneously, the protein starts to deform, when the force reaches a threshold value, the protein molecule suddenly ruptures, and the force on the tip released to zero. (b) As the AFM piezo continues to approach the glass coverslip surface, the tip on glass can not move anymore, as a result, piezo displacement causes tip bending. Therefore, from b to c, the cantilever bending distance = the piezo displacement D (nm); and the direct AFM instrumental electronic signal was measured and recorded for the loading force, F (mv). If we know the cantilever force constant k (pn/nm), then, from the reading of measured f (mv), we can calculate the protein raputure threshold force (pn) =. And this force for protein raputure is significantly smaller than the force calculated based on the piezo displacement distance, where, the force = product of

4 k times d. Because a part of the piezo displacement is contributed from the protein deformation under force, which does not contribute to the actual force loading. S3.2 Control experiments of force spectroscopy. To further prove that the force abrupt drop (47-75 pn drop) recorded in a force curve is due to the HPPK molecule sudden structural rupture, besides the simultaneous identification from the correlated recording of FRET trajectory, we have performed additional control experiments on glass surface that does not have HPPK molecule tethered. All these experiments were done in three different conditions: non-modified bare glass surface, glass surface coated with only -NH 2 terminal ((3-aminopropyl) trimethoxysilane), and glass surface coated with only -CH 3 terminal (isobutyltrimethoxysilane). All the control experiments were conducted in PBS buffer solution (ph=7.4). The pn abrupt force drop seen in the approaching force curves in the correlated measurements were not observed in the approaching force curves recorded in the blank control measurements under all of these three conditions. The results indicate that the pn level force abrupt drop events observed in the approaching curves are only associated with the targeted protein molecules, but not collapse or deconstruction of coated layer molecules. It is also consistent with our finding that the structural rupture under compressive force is a common behavior of protein molecules.

5 Figure S3. Force curves on different glass surfaces. (A) Glass coated with (3-aminopropyl) trimethoxysilane. (B) Glass coated with isobutyltrimethoxysilane. (C) Non-modified bare glass. 4. AFM-FRET correlated nanoscopy. S4.1 AFM-FRET correlated nanoscopy Setup. Our home-built experimental setup consists primarily an inverted optical microscope (Axiovert-200, Zeiss) and an AFM scanning module (PicoSPM, Agilent) in an over-under configuration (Figure S4). The excitation laser (532 nm) beam was reflected by a dichroic beam splitter (z532rdc, Chroma Technology) and focused by a high-numerical-aperture objective (1.3 NA, 100X, Zeiss) on the sample surface at a diffraction limited spot of about 300 nm in

6 diameter. To obtain single-molecule FRET image and photon-counting time trajectories, the emission signal was split using a dichroic beam splitter (640dcxr) into two-color beams centered at 570 nm and 670 nm representing the emissions of the Cy3 and Cy5 donor-acceptor dye pair respectively. The two-channel signals were collected by a pair of Si avalanche photodiode single photon counting modules (SPCM-AQR-16, Perkin Elmer Optoelectronics) for detecting the single-molecule fluorescence. We were able to obtain an fluorescence image (ranging from 1µm 1µm to 100µm 100µm, typically 10µm 10µm) by continuously raster-scanning the sample over the laser focus with an piezoelectric scanning stage (Physik Instruments Inc., Germany) at any scanning speed (typically ranging from 1 ms/pixel to 30 ms/pixel), with each image being normally 100 pixels 100 pixels. Typically, we collect fluorescence intensities of the FRET donor (Cy3) and acceptor (Cy5) for several hundred seconds by a two-channel Picoharp 300 (PicoQuant) time-correlated single photon counting (TCSPC) system. A manual two-axis x-y mechanical positioning stage (Zeiss) and a two-axis close loop x- y 100 µm piezoelectric-scanner stage (Physik Instruments) were mounted directly on the optical microscope. The two-axis close loop x-y piezoelectric-scanner stage was controlled by computer with a raster scan software, by which we were able to scan sample over laser in two-dimension (2D) to provide images and identify positions of dye-labeled single-molecule proteins within the laser focal spot. The two-axis x-y mechanical positioning stage was used to support the closeloop AFM scanning module (PicoSPM, Agilent). With that, we moved the AFM tip in 2D on top of the x-y piezoelectric-scanning stage independently, and positioned the AFM tip to co-axial with the laser beam from the microscope objective. An AFM scanner was used to scan topographic images or manipulate single molecules by force. A home-built fluid cell was put on top of the sample (a transparent glass cover-slide) to keep the sample in buffer solution. To

7 avoid the FRET signal from being interfered by AFM laser, the AFM scanning module was modified by an infrared superluminescent diode (SLD) at 950 nm to replace the conventional 650 nm laser source. A shortpass filter E835sp (OMEGA Optical) was put in front of the detectors to block the AFM infrared photons and a longpass filter HQ545lp (Chroma Technology) was put to block 532 excitation laser: Figure S4. Experimental setup of single-molecule AFM-FRET Nanoscopy.

8 M: Mirror, Dichroic beam splitter 1: z532rdc (Chroma Technology), reflecting 532 nm excitation laser beam and transmitting fluorescence. Dichroic beam splitter 2: 640dcxr (Chroma Technology), splitting the emission signal into two color beams centered at 570 nm and 670 nm representing Cy3 and Cy5 emissions. APD 1: Si avalanche photodiode single photon counting modules (SPCM-AQR-16, Perkin Elmer Optoelectronics) for detecting the single-molecule fluorescence Cy5. APD 2: Si avalanche photodiode single photon counting modules (SPCM- AQR-16, Perkin Elmer Optoelectronics) for detecting the single-molecule fluorescence Cy3. Filter 1: HQ545lp (Chroma Technology), blocking 532 nm excitation laser beam. Filter 2: E835sp (OMEGA Optical), blocking AFM infrared 950 nm beam. For the AFM topological imaging and single-molecule force manipulation, we utilized a typical contact AFM tip coated with Cr/Au to obtain force spectroscopy of the protein underneath. To collect data from one single molecule at a time from both spectroscopic and AFM amplitude channels simultaneously, we spatially lined up the optical focal point and AFM tip precisely so that AFM tip, laser beam focus, and target molecule were in the co-axial alignment. S4.2 Co-axial alignment of the laser and the AFM tip. To line up the optical focal point and AFM tip is the first and critical step for a typical operation of our AFM-FRET Nanoscopy. First, we move the x-y two-axis mechanical positioning stage to roughly align the AFM tip with the laser beam focal point by observing the reflection pattern of the AFM tip; a symmetric light reflection pattern can be observed from the microscope objective. It indicates that the coaxial position is achieved within a few micrometers.

9 To co-axially align the AFM tip with the laser beam center of Gaussian distribution of the laser focus, we scan the AFM tip cross the area of laser beam that has been aligned, and send one of the APD signal to AFM controller through a gated photon counter SR400 (Stanford Instruments, CA) as shown in Figure S5. The image of the optical intensity was taken during AFM tip scanning. Figure S5B shows a bright spot due to the photons from tip reflection as the AFM tip scans over the laser beam, because the tip can be considered as a micro mirror that reflects more photons back through the objective, effectively enhance the photon collection solid angle of the microscope. Through this alignment, we can align AFM tip with the center of laser beam within a hundred nanometers. Figure S5. Schematic diagram of coaxial laser and AFM tip. (A) The AFM tip scans right on the protein and the laser beam focus spot. (B) An optical image of laser focus spot under AFM tip scanning. The bright spot indicates the laser beam position. S4.3 Single molecule measurement and force mapping. After the AFM tip alignment with excitation laser, the reflection is strongest when the apex of the tip is right on top of the molecule. Therefore, by moving the AFM tip to the center

10 of the image, AFM tip, the target single molecule, and the laser focus point are aligned as precise as tens of nanometers. After these procedures, the x-y position of the AFM tip is to be fixed, and moving the sample stage alone can allow us to switch different single molecules and implement further measurements. After the alignment, a sample stage scanning is conducted to look for single protein molecule on the modified glass surface. We can also justify the quality of the alignment by the detected photon counting level of the resulting image. The optical signal from dye labeled single HPPK molecule would be much stronger with the AFM tip right on top of the laser focal point. Even though AFM tip, laser focus and the molecule are coaxially aligned, yet the molecule can be away from the AFM tip by tens of nanometers as the size of the protein molecule is much smaller than the diffraction limit of the fluorescence light. So, to ensure direct contact of the AFM tip apex surface with the molecule, we made a matrix covering nm 2 area under the laser focus where the AFM force curve measurement was taken in every 20 nm interval to ensure a protein is under the AFM tip for a correlated force-manipulation and FRET imaging analysis. As the typical radius of the coated AFM-tip apex is less than 35 nm, and size of the HPPK molecule is only about 5 nm in diameter; therefore, a nm 2 area can be divided in total sixteen 5 5 nm 2 pixels where a protein molecule can be found in any of these pixels. From the figure S6 we can see that protein molecule can be oriented under an AFM tip in four possible ways. Out of those, in two of the cases protein molecule can be compressed without any traces of tensile force. Whereas, in case 1 and 4 there can be very little to negligible amount of tensile force acting on the protein molecule. In this case, it is most likely that the compressive force reaches the protein rupture threshold force value, but the tensile force is still too small to make any impact on the protein except some possible deformation, which may be the reason that we see the broad distribution of the threshold compressive force for the protein

11 ruptures. Nevertheless, force manipulation on a protein molecule represents either one of these four possibilities, making it essentially a compressive force measurement. Figure S6. Force mapping on a single protein molecule (A) Four possible positions of a protein molecule under the AFM tip and the different assessment of the acting force on the protein molecule. (B) Represents the total number of ways a protein molecule can be located under the AFM tip. Green box represents where the protein molecule can be compressed without any trace of tensile force, whereas the blue box represents where the protein molecule can be compressed with very small to negligible amount of tensile force. While the AFM tip is repeatedly pushed down and pulled up, multiple force spectroscopic data and FRET trajectories are collected simultaneously, and they are recorded in the same temporal axis. The FRET efficiency was calculated by the donor acceptor intensity using the formula E FRET = I A /(I A +I D ). Where, I A is the fluorescence intensity of the acceptor and I D is the fluorescence intensity of the donor. It is noticeable that as the AFM tip was moving down and closer to the laser focal point, the detected photon counts in both donor and acceptor

12 channels rose due to a previously reported micro-mirror reflection effect, resulting in a higher signal collection efficiency. Typically, the micro-mirror effect appears or disappears at 150 nm above the sample surface. A B C D Figure S7. The procedures of AFM-focus-point alignment in the AFM-FRET correlated measurement. The black rectangle represents the AFM cantilever. The green circle represents the laser focus point. The size of laser focus in the figure panels are drawn larger than actual size relatively. The red spots represent single protein molecules immobilized on the cover glass.

13 S4.4 FRET intensity trajectories correlated with compressive force curve measurements E FRET Time (ms) Figure S8. Different color represents the ten different FRET intensity trajectories correlated with compressive force curve measurement. (-)ve time represents the time before rupture, (+)ve time represents the time after rupture, and time zero represents the moment of rupture. Black line is the average of all the trajectories with standard deviation error bar. S4.5 Pearson s product-moment correlation coefficient, (, ): Pearson s product-moment correlation coefficient between FRET efficiency and measured force is: r, (T,n)= ( ) ( ( ) )( ( ) ( ) ( ) ( ) ( ) ). T is the index of FRET efficiency time trajectory based on 10ms bin; n is the number of data points within each calculation window, we used 5 in Figure S9B; F(t) is force measured; E(t) is FRET efficiency calculated; F (T),E (T) are the sample averages within each calculation window; and s (T),s (T), are the sample standard deviations within each calculation window, which is defined as s (, ) = ( ) (x(t) x (T)).

14 Similarly, to confirm CaM protein rupture events, we have also plotted the distribution of Pearson s product-moment correlation coefficient between FRET efficiency and force near the force abrupt drop time, showing that the Pearson s product-moment correlation coefficient reaches the maximum at the moment of the rupture events. The distribution of the coefficient appears to be narrowly pin-pointed at rupture event time; whereas the distribution tends to be much broader when t<-0.1 s and t>0.1 s away from the rupture event time. A B Figure S9. (A) Force trajectory of a pushing cycle on a single CaM molecule. Time at the point of rupture considered as time zero, before rupture (-)ve time, after rupture (+)ve time. (B) The distribution of Pearson s product-moment correlation coefficient r F,E (T, n) of force and FRET efficiency near the moment of the CaM protein rupture calculated from 68 sets of AFM-FRET correlated trajectories with 10 ms of binning time. The moment of rupture is redefined as time zero for the trajectories. The black curve is the correlation coefficient trajectory calculated from the trajectory A and its respective FRET efficiency trajectory.

15 Figure S10: Mean of all the thirteen trajectories of Figure 3E, with standard deviation error bar. Blue arrow indicates the point of rupture where the standard deviation error bar is narrow, and the value is close to 1, indicating a high correlation of force drop and the FRET efficiency drop at the protein rupture event. S4.6 Control experiment for the signal-to-noise ratio of the fluorescence photon detection by the correlated single-molecule AFM-FRET nanoscope. After the AFM tip is positioned coaxially with the excitation laser focus, the sample is raster scanned in 10 ms/pixel, and the fluorescence intensity data were collected during the scanning (Figure S11). The signal-to-noise ratio of the fluorescence measurement was calculated by comparing the peak signals (as the fluorescence molecule crossover the scanning laser focal point) and the baseline signal (no fluorescence molecule under the scanning laser focal point).

16 Photon Counts Time (s) Figure S11. One scanning period of the fluorescence intensity-time trajectory for the signal-to-noise ratio control experiment of the AFM-FRET nanoscopy setup. The FRET donor (green) and acceptor (red) channel signals. The baseline is about 10 counts/10 ms, and the peaks signal are mainly distributed in the range from 100 to 150 counts/10 ms. So, the signal-tonoise ratio for the setup is in the range from 10 to Characterization of compressive force. HPPK: To evaluate the relation between threshold force of protein rupture and AFM tip approaching velocity, i.e., the apparent loading rates (ALR), we repeated the experiments under four different ALR. Figure. S12A-D show the statistical distribution of the threshold force at four different ALR. The threshold force required for a single protein HPPK to rupture increases from 47 pn to 75 pn as the approaching velocity increases from 200 nm/s to 2000 nm/s, and the threshold force increases linearly along with log (ALR) (Figure 4B). This dependence of the threshold force upon the loading rate suggests that the protein rupture events and the compressive force loading process have the similar characteristics with a system of two-state conformational transition under an external force.

17 Figure S12. Characterization of HPPK rupture force and loading energy. (A-D) The rupture threshold force distribution at different loading velocities. CaM: We also repeated the experiments for CaM protein under five different ALR. Figure S13 A-E show the statistical distribution of the threshold force at five different ALR. The threshold force required for a single protein CaM to rupture increases from 12pN to 35pN as the

18 approaching velocity increases from 50 nm/s to 1000 nm/s, and the threshold force increases linearly along with log (ALR) (Figure 4D). A B C D E F Figure S13. Characterization of CaM rupture force and loading energy. (A-E) The rupture threshold force distribution in different loading velocities. (F) Distribution of calculated loading energy. Loading velocity is 100 nm/s.

19 We calculated the loading energy using = ( ). is the compressive force loading distance, and F(l) is the loading force curve. Given the force constant of AFM cantilever = 30 pn/nm and the average rupture threshold force is 47 pn under 200 nm/s force loading velocity. In our calculation, we also considered the force being linearly increased during loading process, which gives ( )=. Under the above approximation, the distribution of calculated threshold compressive force loading energy is plotted in Figure 5D (HPPK) and Figure S13F (CaM), given k B T=4.114 pn nm. Most of the rupture events are triggered by loading energy around 30 k B T for HPPK and 4-8 k B T for CaM protein. 6. Additional data. S6.1 Force vs Displacement trace of HPPK rupture. Figure S14. The same force trajectory of Figure 2A (Single HPPK protein rupture) plotted with AFM piezoelectric displacement. S6.2 Single CaM protein rupture data. A B

20 Figure S15. (A) Force vs AFM piezoelectric displacement data of a single CaM protein rupture. (B) Distribution of AFM piezoelectric displacement under 100 nm/s loading velocity, which is defined as the distance traveled by AFM electropiezo between the start of the force loading on a targeted protein and the protein rupture.