Topographical Structure Characterization of Bacteriophytochrome RpBphP2 by
Scanning Tunneling Microscopy
Anna Gawedzka, Stefan Tsonchev, Kenneth T. Nicholson and Emina A. Stojković
Departments of Chemistry and Biology, Northeastern Illinois University, Chicago, IL 60625
Introduction
Structure of the Biliverdin and RpBphP3
Bacteriophytochromes RpBphP2 (P2) and RpBphP3 (P3) are red-light
photoreceptors
isolated
from
the
photosynthetic
bacterium
called
Rhodopseudomonas palustris. Upon light stimulation P2 and P3 mutually direct
synthesis of the light-harvesting complex LH4. P2 like classical
bacteriophytochromes photoconverts between red (Pr) and far-red (Pfr) lightabsorbing states. P3 is unique because it undergoes photoconversion between
red (Pr) and novel near-red (Pnr) light-absorbing state. Pr state is the darkadapted state for both P2 and P3.
Scanning tunneling microscopy (STM) is a novel technology used to analyze
surfaces at an atomic level of metals, semiconductors, as well as molecules
adsorbed on such surfaces Therefore, STM has a variety of applications in many
areas of biology, chemistry, and physics. Gerd Binning and Heinrich Rohrer won a
Nobel Prize in Physics in 1986 for the discovery and development of STM.
Scanning tunneling microscopy uses the principle of quantum tunneling to make
atomic resolution images of the studied sample. The advantage of the STM
technique in surface studies is due to the fact that it is simple to operate, offers
safe experimental conditions and gives high-resolution images.
Topographical structure of bacteriophytochrome P2 was investigated using
STM. Successful imaging of P2 upon red-light illumination was accomplished.
Protein arranged in hexagonal pattern on Highly Oriented Pyrolytic Graphite
(HOPG) surface. The size of the individual P2 molecules in dimer conformation
as determined by STM is 6.59 nm, which directly correlates with the published
crystallographic structure of closely related bacteriophytochrome P3.
Description of the Bacteriophytochrome RpBphP2
A.
A.
STM Images of P2 on the Surface of HOPG
B.
~68 Ǻ
~48 Ǻ
FIGURE 4.
A 40 μL of 0.27 mg/ml P2 protein sample was applied on HOPG substrate and
allowed to dry in air under red-light exposure. These images of protein on
HOPG were acquired at a bias voltage of -50 mV and a tunneling current of
1nA. The areas are 25 x 25 nm2 and 50 x 50 nm2, respectively.
FIGURE 2.
A.
A. Structure of linear tetrapyrrole chromophore biliverdin (BV) found in P2. BV is bounded in
the light-sensing module that comprises of PAS (Per-ARNT-Sim), Gaf (cGMP
phosphodiesterase/adenylyl cyclase/FhIA) and PHY (phytochrome) domain. Red lightinduced isomerization of the C15=C16 double bond is driving changes in the orientation of
the BV and causes conformational change in the protein (2). B. Crystal structure of closley
related dimeric bacteriophytochrome P3. The domains are showed in different colors. Green
color, PAS domain and the red color, GAF domain. Chromophore biliverdin is bounded to
Cys-28 in the GAF domain. Approximate dimensions of the protein measured using Pymol
software were found to be 45 Ǻ and 68 Ǻ (3).
B.
kinase module
Full-length
N
PAS
GAF
PHY
HisKA
CBD+PHY
N
PAS
GAF
PHY
P2 CRYSTALS
ATPase
A.
B.
B
A
A
B
FIGURE 5.
Principles of STM Operation and Experimental Design
photosensory module
B.
A.
A. Hexagonal arrangement of self-assembled monolayer of P2. Six line scans
were used to measure the size of P2 to be 6.16 ± 0.17 nm. Using the
adjustment factor calculated through calibration, P2 has a diameter of 6.59 nm.
The image was scanned at a tunneling current of 1 nA and bias voltage of -50
mV.
B. 3-D view on the protein lattice observed on the HOPG surface.
B.
A
C.
A
0.39
• Characterize the conformational changes in the structure of P2 and P3 upon
red-light exposure using scanning probe techniques
Pr
dark (Pr)
light (Pfr)
B
B
0.19
Future Directions
Pfr
• Hexagonal arrangement of the P2 protein may be driven by remarkable
interaction of protein molecules with each other. STM studies of P2 in the darkadapted state will determine if this pattern is unique to the excited state and
may shed light on electrostatic interactions driving the assembly.
Acknowledgements
FIGURE 1.
-0.01
240
340
440
540
640
740
840
A. Silver-stained denaturing protein gel showing the purity of the isolated P2. After
P2 synthesis was induced in E. coli cultures carrying the genes coding for P2,
column chromatography was employed to separate P2 from other cellular proteins
(lanes 1,3, and 4). B. Domain composition of P2, MW= 76 kD. Domain
arrangement of full-lenght P2 (and P3) showing chromophore binding domain
(CBD) and histidine kinase domain. Here, we imaged P2 protein containing a
photosensory core, PAS, GAF and PHY domains. C. UV-visible absorption spectra
of P2 (PAS-GAF-PHY) lacking histidine kinase domain. Spectra were measured in
solution in the dark-adapted Pr (black) and light-illuminated Pfr (red) states. Pr,
dark-adapted state peak centerd at 710 nm and Pfr, the exciated state centered at
750 nm (1).
FIGURE 3.
A. A probe tip is located at the corner of tripod made from piezoelectric ceramic. The purpose
of the x and y piezoelectric crystals is to move the tip over the sample in the x and y
direction. The z piezoelectric crystal together with the feedback control circuit is used to
monitor the movement of the tip in the z direction maintaining the tunneling current constant
(4). B. Calibration of STM instrument. HOPG imaged upon application of 0.20 mM NaCl. Six
line scans were used to measure the lattice parameter of HOPG to be 0.23 ± 0.01 nm.
Literature reports the value to be 0.246 nm. This derives a correction factor of 1.07. The
HOPG images were acquired at a tunneling current of 1 nA and bias voltage of -50 mV. The
STM measurements were performed at ambient temperature and pressure using the
Nanosurf® easyScan 2 STM.
We would like to thank the Student Center for Science Engagement at NEIU for
financial support. In addition, we would like to thank Dr. Qiti Guo and Dr. Ka Yee
C. Lee at The University of Chicago Materials Research Science and
Engineering Center for helpful discussions and additional training on scanning
probe instruments.
References
1. E. A. Stojkovic. Purification and crystallization of R. palustris RpBphP2 bacteriophytochrome.
Introduction to Laboratory Exercises (2007)
2. E. Giraud et al. Journal of Biological Chemistry. 280:32389-32397(2005)
3. X. Yang, E. A. Stojkovic et al. PNAS. 104:12571-12576 (2007)
4. D. A. Bonnell ed. (1993). Scanning Tunneling Microscopy and Spectroscopy. Theory,
Techniques, and Applications. New York: VCH Publishers, Inc.