MOLECULAR MOTOR RESEARCH GROUP
PROJECT LEADER: ROMAN TUMA, Ph.D.
docent, visiting scientist
tel: 358-9-191-59577
fax: 358-9-191-59920
e-mail: roman.tuma@helsinki.fi
Institute of
Biotechnology
Viikki Biocenter 3 room A2315
PL 65 (Viikinkaari 1)
FIN-00014 University of Helsinki
Finland List of Publications
We have moved to the Astbury
Centre, University of Leeds, UK in Sep
2007, new contact: r.tuma@leeds.ac.uk
RESEARCH
We study assembly and function of
complex biological macromolecular assemblies.The results are
then applied to create new nanometer scale molecular machines and
materials.The research can be divided into four principal areas of
interest:
Virus Assembly and Dynamics
We study assembly and conformational changes in dsRNA bacteriophages of
the Cystoviridae family
(Poranen et al, 2005):
These bacterial viruses are good model systems for the assembly of
other complex dsRNA viruses such as members of the Reoviridae family. We have
developed in vitro assembly systems for several cystoviruses. These
investigations demonstrated the essential role of minor proteins,
namely RNA-dependent RNA polymerase (protein P2) and RNA packaging
motor (protein P4), in the nucleation step of assembly: (Poranen et al,
2001; Kainov et al., 2003).
The structure and dynamics of individual proteins within viral
assemblies can be visualized by hydrogen deuterium exchange (HDX) and
high resolution mass spectrometry (FT-ICR, collaboration with Prof. Alan
Marshall at NHMFL).
Viruses are also good examples of self-assembling structures in
nanoscience starting point for
engineering "smart" self-assembling (bottom-up approach) materials for
nanotechnology. One
example is a combination of a-hemolysin
nanopore
with the
packaging motor P4 which together constitute a device for nucleic acid
translocation across membranes (Colaboration with Dr. Stefan Howorka
at University College London, UK and Prof. Hagan Bayley
at Oxford University).
Selected publications:
M. M. Poranen and R. Tuma (2004). Self-assembly of
double-stranded RNA
bacteriophages. Virus Res. 101, 93-100.
Kainov, D. K., Butcher, S. J., Bamford, D. H. and Tuma, R. (2003)
Conserved
intermediates on the assembly pathway of dsRNA bacteriophages. J.
Mol.
Biol. 328, 791-804.
Poranen, M. M., Paatero, A. O., Tuma, R. and Bamford, D. H.
(2001).
Self-assembly
of a viral molecular machine from purified protein and RNA
constituents. Mol.
Cell. 7, 845-54.
Ikonen, T., Kainov, D. K., Timmins, P., Serimaa, R. and Tuma, R.
(2003)
Locating the minor components of dsRNA bacteriophage phi6
by neutron scattering. J. Appl. Cryst. 36, 525-9.
Mechanisms and Regulation of Viral Molecular Motors
Molecular motors are devices that convert chemical energy (usually
obtained from ATP hydrolysis) into mechanical work and motion. For
biological molecular motors movements range from steps of nanometer or
less (10-9 m) to processive translocations of several micrometers. The
polymerase core of cystoviruses contains two molecular motors:
monomeric RNA polymerase P2 and hexameric packaging motor P4. The
packaging motor translocates ssRNA into preformed capsids at the
expense of ATP hydrolysis and also exhibits helicase activity (Kainov
et al, 2004). It's three-dimensional structure is similar to DnaB
family of hexameric helicases (F4 family). The 3D structure also
revealed conformational changes which may be associated with RNA
translocation (Mancini et al, 2004, Figure on the right, in
collaboration with Dr. Erika Mancini
& Prof. David Stuart, Oxford University).
In addition to X-ray crystallography we employ a combination of
genetic, biochemical and biophysical methods to unravel 
the mechanism of these molecular motors.
These techniques
range from site-directed mutagenesis, chemical crosslinking and
enzymology (Kainov et al. 2004) to stopped-flow fluorescence binding
assays (Lisal & Tuma 2005), hydrogen-deuterium exchange (Lisal et
al., 2005) and single molecule methods. For single molecule
measurements we have constructed in collaboration with the
Electronics Research
Unit at the Department of Physical Science (Prof.
Edward Hæggström)
an optical tweezers instrument (left)
which will be upgraded with single molecule fluorescence detection
system for simultaneous detection of conformational changes. Thus far
these methods demonstrate that P4 hexamer operates as a cooperative Brownian ratchet.
The next step is to visualize RNA binding within the hexamer and look
at the structure and regulation of the motor within the viral
capsid. See our recent publications on single molecule experimental design
and theory (Wallin
et al. (2007) Biophys. J.
doi:10.1529/biophysj.106.097915) and trap steering Wallin et al. (2007) Proc. of
SPIE 66441Y.
Selected publications:
Mancini, E. J., Kainov, D. E., Grimes, J. M., Tuma, R., Bamford,
D. H. & Stuart, D. I. (2004). Atomic Snapshots of an RNA Packaging
Motor Reveal Conformational Changes Linking ATP Hydrolysis to RNA
Translocation. Cell 118, 743-755.
Lísal, J. & Tuma. R. (2005) Cooperative mechanism of
RNA packaging motor. J. Biol. Chem. 280, 23157-64.
Lísal,
J., Lam, T., Kainov, D. E., Emmett, M. R.,
Marshall, A. G., Tuma, R. (2005). Functional visualization of viral
molecular motor by hydrogen-deuterium exchange reveals transient
states. Nat.
Struct. Mol. Biol. 12, 460-466.
Kainov, D. E., Tuma, R.,
& Mancini, E (2006)
Hexameric molecular motors: P4 packaging ATPase unravels the mechanism.
Cell. Mol. Life Sci. 63, 1095-1105.Structure and Assembly of Chlorosomes
Chlorosomes are light harvesting complexes from sulfur and filamentous
green photosynthetic bacteria that are able to survive in extremely
low-light conditions. The chlorosome is a good model for developing
highly efficient light harvesting systems and constitutes promising
biomaterial for the design of solar cells. We use combination of
solution X-ray scattering, cryo-electron microscopy and optical
spectroscopy to gain insight into the arrangement and self-assembly of
pigments (bacteriochlorophylls) within the chlorosome. This is done in
collaboration with Dr.
Jakub Psencik (Charles University, Prague), Dr.
Sarah Butcher (cryo-EM unit, University of Helsinki), and Teemu Ikonen
and Prof. Ritva Serimaa (Department of Physics, University of
Helsinki). Our studies showed that bacteriochlorophyll and carotenoid
pigments self-assemble into lamellar structures, which provide the
framework for fast and efficient energy transfer:
Further structural studies are done on chlorosomes from mutant strains
of Chlorobium tepidum
(collaboration with Prof. Donald Bryant, Penn State, USA) and
chlorosomes from green filamentous bacteria (Chloroflexus aurantiacus).
For further details see: Pšenčík, J., Ikonen, T.P.,
Laurinmäki, P., Merckel, M.C., Butcher, S.J., Serimaa, R.E., Tuma,
R. (2004). Lamellar organization of pigments in chlorosomes, the light
harvesting complexes of green photosynthetic bacteria. Biophys. J. 87, 1165-1172.
Jakub Pšenčík,
Juan B. Arellano, Teemu P. Ikonen, Carles M.
Borrego, Pasi A. Laurinmäki,
Sarah J. Butcher, Ritva E. Serimaa, Roman Tuma
(2006) Internal
structure of chlorosomes from
brown-colored Chlorobium species and the
role of carotenoids in their assembly.
Biophys. J. 91, 1433-40.
Virus Evolution
Structural characterization of a phage PRD1 spike protein
P5 (stereo image on the left) revealed unexpected relationship to
TNF family of signalling
molecules, previously found
exclusively in eukaryotic cells and organisms. This suggests common
evolutionary origin of signalling molecules in the immune system and
viral structural proteins. The study also shed light on the evolution
of viral structure. This work is done in close collaboration with
Prof. Dennis Bamford who
introduced and further developed the idea of structure-based virus
classification.
For further details see: Merckel, M. C., Huiskonen, J. T., Goldman, A.,
Bamford, D.H., Tuma, R. (2005). The structure of the bacteriophage prd1
spike sheds light on the evolution of viral capsid architecture. Mol.
Cell 18, 149-159.
GROUP MEMBERS
Anders Wallin, M.Sc. (graduate student, single molecule biophysics) supported by The Finnish National Graduate School in
Nanoscience
Dr. Ari Ora (posdoctoral fellow shared with Sarah Butcher, virus dynamics,
single molecule biology)Dr. Katarina Hattula (research assistant)
Graduated:
Samuel Atarah, M. Sc. (physics 2001) (M.Sc. Thesis on optical
trapping atarah_2001_progradu.pdf)
Denis Kainov, Ph.D. (genetics 2005),
supported by ISB, thesis,
EMBO fellow at IGBMC, CNRS, Illkirch, France
Jiri Lisal, Ph.D.. (biochemistry 2006), supported by Viikki Graduate
School
of Biosciences,
thesis, HFSP fellow at Stanford University School of Medicine, USA
Jelena Telenius (Medicinal Biochemistry, Dec 2007,
M.Sc. Thesis on molecular motors)
FUNDING
The research group is supported by grants from the Academy of Finland (
Appropriation to academy research fellow 2003-2008 and ESF Euroscope program 2006-2008). The
group participates in and students are supported
by the National graduate school in informational and structural biology
( ISB), Viikki Graduate
School
of Biosciences, and The Finnish
National Graduate School in Nanoscience.