Molecular recognition of super-structured substrates
Our research is focused on the question: How do MMPs interact with their intact substrates like collagen and amyloid fibrils? These fibrilar entities are composed of large and complex protein assemblies. The breakdown of collagen in various pathologies such as inflammatory arthritis and cancer is linked to disease progression accompanied by profound changes in the structure of collagen and its mechanical response. However, the mechanism and directionality of collagen degradation by collagenases, members of the MMP family, remains an enigma.
We want to understand effective degradation of collagen molecules by investigating the specific modes of interaction and alteration of enzymes with the ultra structure of monomeric and fibrillar collagen which lead to degradation or remodeling.
Single-molecule imaging of enzymes and collagen molecules by atomic force microscopy allows us to observe directly how individual MMPs, possessing relatively small active sites, bind and degrade large molecular assemblies such as triple helical collagen molecules.
Do water dynamics contribute to metalloenzyme function?
The tendency of hydrophobic surfaces to aggregate in water is often invoked to explain how biomolecules recognize and bind to each other. Water seems to have a much more active role in these processes than had been thought.When biomolecules interact, what do the surrounding water molecules do? One might think that their job would simply be to get out of the way, a crowd that must stand aside for the main actors. But there is now good reason to believe that water has a much more active role in the dialogue between the more celebrated constituents of the cell. When a protein binds its ligand, associates with another protein or folds into its functional form, the surrounding solvent acts as a versatile intermediary and facilitator. Combining X-ray and Thz (IR-based) spectrocopies we uncovered the subtlety and sophistication of that role and, in doing so, challenge some common perceptions of how biomolecular recognition operates within an enzyme cavity.
The structure-function relationship of matrix-metalloproteinases (MMPs) has been studied extensively, while the influence of the few hundred to thousand water molecules fluctuating around each MMP during enzymatic reactions has been experimentally out of reach. By combining real-time spectroscopic and stopped-flow techniques, we detected the collective interplay of enzyme-substrate interactions and water dynamics of the enzyme membrane type-1 matrix metalloproteinase (MT1-MMP).
X-ray absorption spectroscopy in real-time focused on the enzyme active site charge-fluctuations and structural-kinetic events at atomic resolution. Water network dynamics around MT1-MMP were detected by kinetic terahertz absorption (KITA) which is specifically sensitive to collective dynamics of hydration water molecules around proteins while a reaction is proceeding.
In the absence of substrate molecules, a steep gradient of fast to slow water motions from bulk water towards the active site of MT1-MMP exists which is perturbed upon formation of a Michaelis complex of enzyme and substrate. During substrate binding and hydrolysis, specific structural dynamics at the active site occur which are accompanied by charge fluctuations at the catalytic zinc ion. Variations of the zinc charge may induce fluctuations of polarization into the water network thus facilitating binding and conformational docking of substrates molecules which are then hydrolyzed at the enzyme active site. In any event, molecular recognition here is much more than a case of complementarity between receptor and substratesubstrate - it also crucially involves the solvent. This suggests that changes in protein and solvent dynamics are not mere epiphenomena, but have a vital role in substrate binding and recognition: they are more cause than consequence. Published in Grossman et al, Nature SMB 2011 and highlighted in Nature 2011 by P. Ball. Based on these findings we are now up to advance our understanding of the molecular interplay of enzyme and water dynamics with regard to enzyme functional events.
Role of water in biomineralization
Biomineralization dictates the development of our cartilage and bones, the fixation of carbon dioxide in the deep of the oceans and the growth of various geological structures on the earth`s crust. Biominerals are distinct in their morphology giving rise to the question, which factors determine their fate resulting in a rough or smooth surface, or a structurally dense or sparse material. Fine-tuning of these motifs occurs in water, yet the molecular mechanism of biomineralization under the influence of the dielectric and polar medium remains enigmatic.
We apply X-ray scattering and terahertz spectroscopy to address the challenge of biomineral growth from two perspectives simultaneously. Biomineral growth kinetics is directly monitored by X-ray scattering whereas the collective dynamics of the water network are detected by terahertz absorption spectroscopy. Merging both techniques will elucidate the functional interplay of water dynamics and the growing biomineral in real time.