In order to understand how protein molecules operate in bio-systems such as membrane or cells, some X-ray techniques were developed to give the t i m e - a n d - s p a c e a v e r a g e d s t r u c t u r a l i n f o r m a t i o n a b o u t p r o t e i n m o l e c u l e s . R e c e n t l y , t h e d y n a m i c b e h a v i o r o f s i n g l e b i o - m o l e c u l e s w e r e d i r e c t l y visualized in real time under an optical microscope [1,2]. These single-molecule techniques have been pr ov id in g po si ti on al in fo rm at io n at an ac cu ra cy of ab ou t λ / 10 0, fa r be lo w th e op ti ca l di ff ra ct io n li mi t ( ~ λ / 2 ) . I n t h i s w o r k , w e d e m o n s t r a t e d t h e d i r e c t observations of the rotating motion of an individual single nanocrystal, which is bound to individual bio- m o l e c u l e s , u s i n g t i m e - r e s o l v e d L a u e d i f f r a c t i o n . We ac hi ev ed ti me -r es ol ve d X- ra y ( λ X- ra y ~0 .1 nm ) ob se rv at io ns of pi co me te r- sc al e ( λ X- ra y / 10 0) sl ow B r o w n i a n m o t i o n i n i n d i v i d u a l b i o - m o l e c u l e s i n various aqueous solutions. M o s t X - r a y e x p e r i m e n t s a r e b a s e d o n t h e average of several observations of many molecules a n d t h e b e h a v i o r o f e a c h m o l e c u l e c a n n o t b e d e t e r m i n e d . I n t h i s r e p o r t , w e p r o p o s e d a n e w Dynamical Observations of Individual Protein Molecules with X-rays X - r a y m e t h o d o l o g y f o r d i r e c t o b s e r v a t i o n s o f t h e behavior of single molecular units in real time and in real space. The new system ( Fig. 1 ), which we cal l Dif fra cte d X-r ay Tra cki ng ( DXT ), mon ito rs the B r o w n i a n m o t i o n s o f a s i n g l e m o l e c u l a r u n i t b y o b s e r v a t i o n s o f X - r a y d i f f r a c t e d s p o t s f r o m a n a n o c r y s t a l , t i g h t l y b o u n d t o a n i n d i v i d u a l s i n g l e molecular unit in bio-systems [3,4]. DXT does not d e t e r m i n e a n y t r a n s l a t i o n a l m o v e m e n t s , b u t o n l y or ie nt at io na l mo ve me nt . He re , in or de r to de te ct i n t r a - m o l e c u l a r B r o w n i a n m o t i o n s i n i n d i v i d u a l si ng le bi ol og ic al mo le cu le s on a pi co me te r sc al e, w e u t i l i z e d i n d i v i d u a l d i f f r a c t i o n s p o t s f r o m a n a n o c r y s t a l , w h i c h w a s t i g h t l y b o u n d t o t h e D N A molecules under observation ( Fig. 2 ). We used the white X-ray mode ( Laue mode) of the RIKEN Structural Biology II beamline BL44B2 t o r e c o r d L a u e d i f f r a c t i o n s p o t s f r o m A u n a n o c r y s t a l s . T h e p h o t o n f l u x a t t h e s a m p l e p o s i t i o n w a s estimated to be about 10 15 photon/sec/ mm 2 in the energy range of 7 - 30 kV. The X-ray beam’ s focal size was 0.2 mm (horizontal) × 0.2 mm (vertical). A diffraction spot was monitored with an X-ray image intensifier (Hamamatsu Photonics, V5445P ) and a C C D c a m e r a ( H a m a m a t s u P h o t o n i c s , C 4 8 8 0 - 8 2 ) with 656 × 494 pixels. The average exposure time Fig . 1. Sch ema tic dra win g of the det ect ion sys tem for sin gle mol ecu lar tra cki ng with X-rays (not to scale). Diffracted X-ray Tracking ( DXT ) monitors the motion of a s i n g l e n a n o c r y s t a l w i t h t h e g u i d a n c e o f a d i f f r a c t i o n s p o t f r o m t h e i n d i v i d u a l nanocrystal itself, which is labeled with the individual single molecular unit. Laue Diffraction Substrate White X-rays 23 2 θ = 415 mrad 2 θ = 345 mrad 2 θ = 296 mrad 2 θ = 271 mrad F i g . 2 . S c h e m a t i c d r a w i n g o f t h e X - r a y s i n g l e - m o l e c u l a r d e t e c t i o n s y s t e m f o r i n d i v i d u a l D N A m o l e c u l e s i n a q u e o u s s o l u t i o n s ( n o t t o s c a l e ) . T h e d i a m e t e r o f t h e n a n o c r y s t a l a n d t h e D N A m o l e c u l e are about 15 nm and 2.5 - 3 nm, respectively. was 1 msec. Figure 3 shows movements of diffraction angles θ from a single nanocrystal coupled to a single DNA m o l e c u l e ( 1 8 - m e r ) a t 4 C . T h e o b s e r v e d s p o t s r a n d o m l y m o v e a l o n g t h e d i r e c t i o n o f θ . F r o m an al yz ed da ta , th e ob se rv ed di sp la ce me nt of θ is assigned as directed Brownian motion [5]. In th e fu tu re , th e bi gg es t ch al le ng e wi ll be to observe individual and rare biological processes in l i v i n g c e l l s . D X T c a n b e u s e d t o m o n i t o r n o t translational motions but orientational ones on pico- meter scales. DXT can be expected to observe the stru ctur al chan ges acco mpan ying the acti vati on of i o n c h a n n e l s i n l i v i n g c e l l s . S u c h c h a n g e s a r e known as tilting or small orientational motions of the helix in channel pores. DXT can also be expected t o m o n i t o r t h e d y n a m i c s o f i o n c h a n n e l s t h r o u g h i o n i c f l u x m e a s u r e m e n t s b y t h e p a t c h - c l a m p technique. References [ 1 ] T h . B a s c h e e t a l . , S i n g l e M o l e c u l e O p t i c a l Detection, Imaging, and Spectroscopy ( Wiley-VCH, Munich, 1997). [2] S. Weiss, Science 283 (1999) 1676. [ 3 ] Y . C . S a s a k i e t a l . , P h y s . R e v . E 6 2 ( 2 0 0 0 ) 3843. [4] Y. C. Sasaki, Y. Okumura, S. Adachi, Y. Suzuki, N . Y a g i , N u c l . I n s t r u m . M e t h . A 4 6 7 - 4 6 8 ( 2 0 0 1 ) 1049. [5] Y. C. Sasaki, Y. Okumura, S. Adachi, N. Yagi, Phys. Rev. Lett. 87 (2001) 248102. Diffracted X-rays DNA Substrate Nanocrystal Yuji C. Sasaki a , b , c (a) SPring-8 / JASRI (b) Osaka University (c) Japan Science and Technology Corpotation, CREST E-mail: ycsasaki @ spring8.or.jp F i g . 3 . E x a m p l e s o f t h e d i f f r a c t e d s p o t s f r o m t h e s i n g l e n a n o c r y s t a l i n a q u e o u s solutions appeared as brightly shining dots (white-blue). Frames are spaced at 180-ms intervals. The exposure time was 1 s. ° 24
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