OUR RESEARCH

All biological processes are executed through a sophisticated web of biomolecular interactions. Learning how to pull the strings of this web with a high degree of selectivity by manipulating certain interactions will provide enormous benefit to all life sciences and especially to molecular therapeutics. However, it requires better tools to study biomolecular structure, dynamics and interactions in complex systems

In the post-genomic era, the spectacular successes of proteomics and bioinformatics have resulted in an explosive growth of information on the composition of complex networks of proteins interacting at the cellular level and beyond. Coupled with the avalanche of new high-resolution protein structures, these advances raise hopes that rational manipulation of these networks aimed at achieving a desired therapeutic outcome will soon become possible. However, a simple inventory of interacting proteins and the availability of their structures are often insufficient for understanding how the components of sophisticated biological machinery work together. It is becoming increasingly clear that the multiplicity of protein conformations, as well as transitions among them, are critical determinants of protein-protein interactions. Contrary to earlier beliefs, conformational heterogeneity and large-scale dynamics are important and vital characteristics of such interactions. Indeed, the results of our own research emphasize the importance of structural plasticity both in modulating functional properties and assembly of multi-unit proteins.

Above and beyond protein-protein interactions, a variety of other biopolymers participate in forming critical nodes within the sophisticated interactomes. In addition to protein-oligonucleotide binding events playing obviously important roles at the terminal points in such networks (e.g., gene expression), a variety of other interactions provide important mechanisms to transmit, suppress or modify the signals both inside and outside the cell. These include interactions involving non-coding RNA and glucosaminoglycans to name a few. Furthermore, the emergence and rapid progress of macromolecular therapeutics and nano-medicine brings to the fore the question of how biomolecules interact with abiotic macromolecules, such as polymers and functionalized nanoparticles.

The central role of macromolecular interactions in fields as diverse as biophysics, structural biology and nanotechnology places a premium on the ability to characterize them. However, experimental investigation of architecture and conformational heterogeneity of proteins, as well as their associations with each other, remains a very challenging task. Characterization of higher order structure and dynamics of other biopolymers, particularly those whose synthesis is not genetically controlled, is even more challenging. One particularly unforgiving limitation inherent to almost all experimental techniques used to probe macromolecular structure and dynamics is the extreme difficulty in characterizing behavior of individual biopolymers in multi-component systems, which arises due to inevitable signal interference from different species.

What unique information does biological mass spectrometry provide on architecture, dynamics and interaction of biopolymers?

Mass spectrometry (MS) has emerged relatively recently as an attractive alternative in the studies of protein architecture and dynamics, capable of providing information on protein conformation at various levels. It also has a tremendous potential for probing higher order structure of other biopolymers, which is yet to be fully explored.Electrospray ionization (ESI) MS provides a means to desorb intact biopolymers (proteins, oligonucleotides, polysaccharides, etc.) from solution to the gas phase. In many cases it is even possible to preserve non-covalent biomolecular complexes and thus obtain information on binding properties in solution (e.g., protein quaternary structure, composition of protein-ligand complexes, etc.). ESI MS is also unique in its ability to detect distinct protein conformers that may co-exist in solution under equilibrium. Concentration requirements are usually very modest, which in many cases allows the biomolecular behavior to be studied at (or even below) the endogenous levels. Importantly, ESI MS is capable of carrying out measurements in complex mixtures, where distinction among various species is made based upon the differences in their masses.

Mass spectrometry-based experimental tools developed in our laboratory

One of the focal points of our research efforts is developing novel mass spectrometry-based strategies to study protein architecture and dynamics. One of such strategies utilizes chemometric tools to detect and characterize multiple protein conformers in solution. Dynamics and structure of these states is probed by a combination of protein chemistry in solution (hydrogen/deuterium exchange to label dynamic segments within the protein) and in the gas phase (protein ion fragmentation to measure the deuterium content across the protein sequence). The latter becomes possible due to a rapid progress in ion fragmentation techniques, which allow primary structure of large biopolymers to be determined in a single experiment. One of our ultimate goals is to use ESI MS to model in vivo processes that are already exploited in medicine or show significant promise as therapeutic targets.

You can learn more on how we use ESI MS to study behavior of specific biopolymers by browsing through our publications or clicking one of the following links:

· Mass spectrometry reveals the secrets of protein interaction with small ligands: retinoic acid binding to its intracellular transporters

· Mass spectrometry provides details of iron interaction with transferrin: mechanistic studies of metal delivery to cells

· Disorder as a molecular lubricant: surprising details of multi-unit protein assembly revealed by ESI MS

· Two-dimensional ESI MS analysis clarifies the link between small-scale conformational transitions and enzymatic activity of pepsin

· ESI MS and the grand challenge of structural biology: protein interaction with highly heterogeneous targets