Prof. dr hab. Wojciech Rypniewski
Head of the Structure-Function Relationship in Biological Molecules
Group at the Center for
Biocrystallographic Research at the Institute of Bioorganic Chemistry
of the Polish Academy of Sciences in
- B.Sc. (Hons) 1983: University of York, England.
- Ph.D. 1986: Cambridge University, England.
- Habilitation 2001: Institute of Bioorganic Chemistry, Polish Academy of
- Professor of chemistry 2007: entitled by the President Lech Kaczynski.
Structure and function of biological molecules by X-ray diffraction
The three-dimensional structure of biomolecules is essential in
determining their function. One of the best tools for studying structure is
X-ray crystallography. My group uses this technique to investigate the
relationship between structure and function for proteins and nucleic acids.
There are several areas under investigation in the laboratory.
Enzymes of key significance in metabolism
Glycolysis is the foundation of both
anaerobic and aerobic respiration and occurs in nearly all organisms. It is
the main energy source in many prokaryotes and in the eukaryotic cell types
devoid of mitochondria or functioning under low oxygen or anaerobic
conditions. The rate of glycolysis is tightly regulated subject to the
cell's needs for energy and building blocks for biosynthetic reactions.
Phosphofructokinase (PFK) is a key control point in the glycolytic pathway,
just downstream of the entry point for hexose sugars. It catalyses the
conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and ATP to
ADP. PFK is the enzyme with the most complex regulatory mechanism in the
glycolytic pathway. The major isozyme of PFK is PFK1, a multi-subunit
allosteric enzyme whose activity is modulated by a number of effectors. The
sophistication of the control mechanism of PFK in eukaryotes is matched by
its complex evolutionary history. Subunits of eukaryotic PFKs are a result
of tandem gene duplication of the prokaryotic precursor with the redundant
parts having evolved to acquire new functionalities, allowing the enzyme to
become responsive to an even larger range of allosteric effectors than the
We have been studying the crystal structures of two eukaryotic PFKs: from
baker's yeast (Saccharomyces cerevisiae) (shown) and from the
skeletal muscle of rabbit, in complex with their ligands to determine the
structure-function relationship of this key glycolytic enzyme: the
enzymatic reaction mechanism and the allosteric control, and to understand
the evolution of this complex enzyme in terms of the 3D structure. The
control of metabolic pathways in eukaryotic micro-organisms, in particular
fungi, has a major impact on economy and medicine. Yeast is ubiquitous in
nature and is used on a large scale in industry (e.g. in fermentation of
sugar) and in biotechnology (e.g. as eukaryotic systems for protein
expression), and for the production of organic compounds - semiproducts in
the synthesis of therapeutics. Some species of yeast are pathogenic.
Mutations in human PFKs have been linked to several genetic diseases.
RNA fragments relevant to pathogenesis and control processes in
"Molecular biology is undergoing its biggest
shake-up in 50 years, as a hitherto little-regarded chemical called RNA
acquires an unsuspected significance" (The Economist). This is due to the
recent discoveries that in addition to the traditionally recognised roles
in ferrying the genetic code across the nuclear membrane and in protein
synthesis, RNA also plays major roles in biological processes which had not
been ascribed to it previously: the catalysis and regulation of gene
expression (miRNAs and siRNAs). There is also a growing interest in
applying RNA technologies in therapeutics (ribozymes, RNAi). RNA possesses
structural richness that matches its newly discovered functions and the
knowledge of its structure, like with other types of biological molecules,
is the key to understanding its properties, its function and interactions
with the environment. Yet RNA is by far the least studied type of
macromolecule in terms of three-dimensional structures.
We carry out crystallographic studies of physiologically relevant
non-canonical RNA duplexes, including their interactions with solvent and
small ligands, and RNA fragments that undergo hairpin-duplex transition.
Recently, have become very interested in RNA sequences containing CNG
repeats (where N stands for any nucleotide). Such structures are implicated
in about 20 neurological diseases. In these studies we collaborate with our
colleagues from the Laboratory of Structural Chemistry of Nucleic Acids,
the Laboratory of RNA Chemistry and the Laboratory of Cancer Genetics
Ultra-high resolution studies of biological molecules
Another area of our interest are the fundamental properties of proteins
interactions with ligands and substrates. We have done a series of atomic
resolution studies of serine proteases and protease/inhibitor complexes.
Atomic resolution (ca. 1 angstroem) data obtained using synchrotron
radiation reveal information previously inaccessible to crystallographers.
Whereas conventional crystallography yields little more than the fold and
approximate disposition of residues, with high resolution studies it is
possible to examine in detail the anisotropic thermal motions of atoms,
visualise hydrogen atoms (left), the details of protein hydration
and the solvent structure. Unrestrained (unbiased) refinement of atomic
coordinates results in molecular models accurate enough to address issues
like differences in substrate specificity and detailed reaction mechanisms.
At resolution much higher than 1 angstroem it is possible to examine the
detailed electronic structure of the molecule and observe features such as
bonding electrons and free-electron pairs (right).