Our research addresses relevant biological questions involving molecular recognition, protein folding and stability. We are currently studying the role of cytosolic human thioredoxin in cancer cells, the structural biology of immunologically active protein fragments, the electrostatic contributions to protein stability, the mechanism of oligomerization, and natively unstructured proteins. Our approach is interdisciplinary and involves:
- The use of complementary tools from molecular and cell biology, microbiology, protein chemistry, biochemistry, and biophysics.
- Calorimetry and spectroscopies such as fluorescence, circular dichroism, and nuclear magnetic resonance constitute the backbone of our
biophysical studies of proteins and protein fragments.
Human cytosolic thioredoxin (Trx-1) is a 12 KDa multifunctional α/β protein which works in concert with
thioredoxin reductase (TrxR) and NADPH as a reductase of many intracellular proteins and a scavenger of reactive oxygen
species (ROS). Some transformed cell lines overexpress Trx-1 and show resistance to anticancer drugs. Histone
deacetylase inhibitors (HDACi) are promising anticancer drugs which cause cell death in certain transformed but not
normal cell lines. The increased level of Trx-1 expression in those normal cell lines plays an important role in their
resistance toward HDACi induced cell death, raising the question of why those normal but not the transformed
cells respond to HDACi exposure by increasing the Trx-1 levels and developing resistance toward HDACi induced cell
death. Characterization of the myriad of intracellular interactions involving Trx-1 is important to identify targets of
opportunity for early cancer screening and rational development of cancer therapies.
The objective is to use Trx-1 fused to a green fluorescent protein (GFP) as an intracellular biosensor to detect the
response of normal and transformed human cells when exposed to promising anticancer drugs. The long term goal
is to understand the mechanism of action of cytosolic human thioredoxin in normal and transformed human
cells.
These are collaborative studies with molecular and cell biologists.
Support from Memorial Sloan-Kettering Cancer Center/City College of New York Partnership.
In contrast to the full-length cytosolic human thioredoxin, a natural N-terminal fragment of 80 aminoacids (HuTrx80)
exhibits an unexpected mitogenic cytokine activity. In close collaboration with computational biophysicists,
computational simulations and complementary biophysical/biochemistry experiments are currently being conducted on HuTrx80 to provide
a structural context for understanding the molecular mechanisms underlying its mitogenic activity. Insight into these
mechanisms has potential for revealing new therapeutic strategies in processes ranging from inflammatory response to
defense against infectious agents.
Current funding is provided by a CUNY Collaborative Award with
Prof. Marco Ceruso.
Electrostatics is central to the relationship between structure and function of proteins. Experimental and theoretical
studies of electrostatics in the folded state have advanced our understanding, but more studies of the unfolded state
are needed to calculate the biophysical properties of proteins. The scarcity of direct measurements and the need to
extrapolate from indirect measurements in the unfolded state have resulted in controversial pKa values of
ionizable residues in that state. Improvements of models for the pH-dependence of the unfolding ΔG in proteins
require these pKa values and also ways to include contributions from interacting ionizable residues in the
folded state. To resolve these issues, direct NMR and CD measurements of electrostatics contributions in both unfolded
and folded states will be conducted using heterodimers in equilibrium with unfolded monomers. Pairs of isolated
disordered complementary fragments of a human thioredoxin (Trx) variant, a well studied representative of Trx
superfamily with known pKa values have been chosen due to their ability to reassemble into native-like
heterodimers upon recombination. This brings together the expertise of computational biophysicists to
calculate electrostatics in the folded and unfolded states of proteins and our expertise in the biophysical
characterization of natively disordered protein fragments. The long term goal is to address the
following question: How do the individual ionizable residues modulate protein stability? To make progress towards this
goal, our studies will be organized around the following specific aims: (1) to test whether the pKa 's values
of ionizable residues in host-guest tetrapeptides are representative of the pKa 's in the unfolded state of
proteins; (2) to test whether the "zero interaction model" accounts for the pH-dependence of the unfolding ΔG of
a protein whose ionizable residues are independent from each other; and (3) to determine how the conserved triad of
carboxylates from the Trx superfamily modulate the pKa value of the individual carboxylates in the folded and
unfolded.
Current funding is provided by a National Science Foundation Award from August 2005 till August 2009.
Biophysical Studies of Oligomerization
Advances in computational tools and the availability of sequences from entire genes have opened the new field of
structural genomics. 3D-structure prediction of protein oligomers is still a challenge for computational
biochemists. Understanding the mechanism of molecular recognition in oligomerization should assist in completing the
structural data base of proteins and their assemblies. Oligomers are found in phage assembly, inclusion bodies,
amyloids etc. Establishing their mechanism of formation is essential to modulate these processes. The fragments of
oxidized E. coli thioredoxin (Trx), a single domain protein of 108 residues, which has been well studied by fragment
complementation, provide a unique opportunity to study oligomerization. In close collaboration with
Prof. Tatyana Polenova,
solid-state nuclear magnetic resonance (SSNMR) methodologies, and complementary biochemical
and biophysical techniques will be used to establish the principles underlying oligomerization and the characterization
of interfaces in heterodimers and homodimers.
Learning About Natively Unstructured Proteins
We have determined the regions of the sequence which drive the folding of thioredoxins (E. coli and human) using protein fragment
complementation,
stopped flow experiments,
differential scanning calorimetry,
fluorescence, circular dichroism, and
NMR spectroscopies.
Our studies indicate that the antiparallel packing of the hydrophobic central β-strands βi (yellow) and
βi+2 (pink) constitutes a folding nuclei. Based on that finding, we have engineered heterodimeric
reassemblies of Trx around different interfaces by complementation of its intrinsically unstructured fragments. These heterodimers
constitute excellent model systems to unravel the mechanism of molecular recognition involving the increasing number of natively
unstructured proteins undergoing coupled folding/binding events in living cells.
Our studies combine three areas of expertise (protein fragment complementation, calorimetry of proteins, and structural NMR analysis of
proteins) and center on two main hypotheses:
- Isolated fragments which comprise the region of only one of the hydrophobic central β-strands (βi (yellow) and
βi+2 (pink)) remain intrinsically unstructured.
- The differences in ΔG of folding/binding among the different heterodimeric reassemblies are mainly due to differences in the
non local interactions within the isolated intrinsically unstructured protein fragments.