We have successfully observed dynamical Brownian motions in an individual protein molecule and other biological ones in real-time with one-hundredth the atomic-scale precision (picometer-scale precision) using X-rays of the super photon ring-8 (SPring-8).

Introduction

All X-ray techniques are based on the averaged measurements of many molecules or atoms. Thus the behaviours of individual single protein molecules cannot be determined using X-rays. In this work, we demonstrated the direct observations of the rotating motions of the individual single nanocrystal, which is linked to the individual specific sites in single protein molecules, using time-resolved Laue diffraction technique.

In visible light wavelength λ, in vivo observations have greatly progressed due to the remarkable development of fluorescence single-molecule detection techniques [1,2]. These single-molecular techniques have provided positional information at an accuracy of about λ/100, far below the optical diffraction limit (i.e. ≈λ/2). Now, we have achieved time-resolved X-ray (λX-ray≈0.1 nm) observations of picometer-scale (λX-ray/100) slow Brownian motions of individual protein molecules in aqueous solution. This single molecular detection system, which we call DXT (diffracted X-ray tracking), was used to monitor the local Brownian motions of individual molecules, as shown in Figure 1 [35].

Schematic drawing of arrangements for single molecular tracking with X-rays (not to scale)

Figure 1
Schematic drawing of arrangements for single molecular tracking with X-rays (not to scale)

DXT monitors motions of a single nanocrystal with the guidance of a diffraction spot from the individual nanocrystal itself, which is labelled with the individual single molecular unit. For example, we observed dynamical single-molecule observations of DNA, actin filaments and myosin heads.

Figure 1
Schematic drawing of arrangements for single molecular tracking with X-rays (not to scale)

DXT monitors motions of a single nanocrystal with the guidance of a diffraction spot from the individual nanocrystal itself, which is labelled with the individual single molecular unit. For example, we observed dynamical single-molecule observations of DNA, actin filaments and myosin heads.

In order to control the number of the binding sites on the labelled gold nanocrystals, we utilized both the periodical structure in actin filaments and the mercury compound. There are cysteine sites at 5.5 nm intervals with individual actin filaments. The diameter of the gold nanocrystal is approx. 15–20 nm. Thus the number of the binding sites can be controlled for either single or double binding using the mercury compound. As a second example of DXT measurement, we observed the slow Brownian motions of the individual lever arm domain within the myosin S1 (subfragment-1) molecule.

Experimental

Using the DXT method, it is possible to monitor the Brownian motions of adsorbed molecules or particles. Since the structure of the actin filament is helical, the flexibility of actin filaments is independent of the adsorbed orientation, which is parallel to the central axis of actin filament. Figure 1 shows a cross-sectional view of the adsorbed actin filament. To adsorb actin filament on to an amorphous gold substrate, we utilized the interaction between the reactive amino residue (lysine, serine and tyrosine) in actin and the amorphous gold surface with a cross-linking reagent, LC-SPDP {succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate; Pierce Co. Ltd) in 50 mM Hepes, pH 8.0, for 6 h at 25°C.

Gold nanocrystals were fabricated using a sequential process. First, the gold elements were evaporated at a thin film thickness (=10 nm) on the NaCl (100) substrate under vacuum conditions. We confirmed that the diameter of the gold nanocrystal was 20–30 nm by scanning electron microscopy (S-5000; Hitachi Ltd). In order to disperse the evaporated gold in the aqueous solutions without any aggregation, the surface of the substrate was dissolved in the detergent solution containing 50 mM CHAPS, pH 7.0.

In order to adsorb the histidine-tagged myosin S1 molecule on to the quartz substrate, the Ni-NTA (Ni2+-nitrilotriacetate) chelating ligand [6] was coupled to the surface of the quartz substrate. The mutant myosin S1 droplet was mounted on the surface of the Ni-NTA ligands, and reacted for 2 h at 4°C. After the adsorption reaction of S1 was stopped, the surface of adsorbed myosin S1 molecules was rinsed with solution [50 mM Tris/HCl (pH 8.0)]. Finally, 50 μl of this same solution containing the nanocrystal was mounted on the adsorbed myosin molecules, and reacted for 2 h at 4°C. In order to bind the C-terminus of the lever arm domain with the nanocrystal, we used gold nanocrystals.

We used the white X-ray mode (Laue mode) of beam-line BL44B2 (RIKEN Structural Biology II; SPring-8, Japan) to record Laue diffraction spots from nanocrystals. Photon flux at the sample position is estimated to be approx. 1015 photon/s per mm2 in the energy range from 7–30 kV. The X-ray focal beam size is 0.1 mm (horizontal)×0.1 mm (vertical). A diffraction spot was monitored with an X-ray image intensifier (Hamamatsu Photonics; V5445P) and a CCD camera (Hamamatsu Photonics; C4880-82) with 656×494 pixels as shown in Figure 2. The thickness of the solution on the substrate (≈7 μm) was controlled with the thickness of the spacer between the substrate and the polyimide film.

A photograph of the instrumental arrangements for DXT

Results and discussion

We could not detect the displacement (Δ2θ≈400 mrad/36 ms) of the observed diffraction spots because of the size and characteristics of the X-ray detection camera. Thus the gold nanocrystals that were not labelled with the actin filament were not detected by this DXT system. Thus the presence of the detectable moving spots from the individual nanocrystals represents the interaction between the nanocrystals and the actin filament or myosin S1 molecules.

In order to characterize the motions of the individual labelled nanocrystals accurately, we utilized a method based on plots of the mean-square displacement against the time interval. In two-dimensional motions, D=〈Δz2〉/(4Δt), where D is the two-dimensional diffusion coefficient, 〈Δz2〉 is the two-dimensional mean-square displacement for time-lags Δt=(ti−tj), 〈Δz2〉=(1/Σ)Σ[z(ti)z(tj)]2, and z(ti) represents the position of the molecule at time ti. In DXT, the relationship between the displacement of the observed diffraction angles Δθ and 〈Δz2〉 is 〈Δθ2〉d2=〈Δz2〉, where d represents the distance between the C-terminus of F-actin and the rotating centre (the surface of the gold substrate). Therefore the values of Δθ from our DXT can be converted into values of Δz.

In dynamical observations of actin filament, we obtained 〈Δθ2〉−Δt curves of labelled gold nanocrystal using a mercury compound (p-chloromercuribenzoic acid) under a Mg2+ aqueous solution with or without the presence of phalloidin. We observed that the displacements (〈Δθ2〉−Δt, Dnon-phalloidin=0.18±0.08 mrad2/s) of the observed spots without the presence of phalloidin were greater than those (Dphalloidin=0.058±0.01 mrad2/s) where phalloidin is present. This result means the rigidity of actin filaments is increased in the presence of phalloidin, and this is in reasonable agreement with many other experimental results [7].

In myosin S1 molecules, the observed motion of the lever arm domain in the myosin S1 molecules is assigned as Brownian random motion during the observed times (≈1 s); nevertheless, the myosin molecules are adsorbed on the quartz substrate through the linear chain molecules. Next, with a solution comprising the molecule ATP (1 mM), 〈Δθ2〉−Δt plots show that the displacement is contained within a limited area and cannot move out of the area during the observation period (≈1 s). The result shows that the Brownian motion of the lever arm domain is spatially corralled by binding the Mg2+-ATP molecule.

Structure Related to Function: Molecules and Cells: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by D. Alessi (Dundee, U.K.), T. Cass (Imperial College London, U.K.), T. Corfield (Bristol, U.K.), M. Cousin (Edinburgh, U.K.), A. Entwistle (Ludwig Institute for Cancer Research, London, U.K.), I. Fearnley (Cambridge, U.K.), P. Haris (De Montfort, Leicester, U.K.), J. Mayer (Nottingham, U.K.) and M. Tuite (Canterbury, U.K.).

Abbreviations

     
  • DXT

    diffracted X-ray tracking

  •  
  • Ni-NTA

    Ni2+-nitrilotriacetate

  •  
  • S1

    subfragment-1

  •  
  • SPring-8

    super photon ring-8

The synchrotron radiation experiments were performed at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (proposal no. 2002B0019-NL2-np).

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