Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions

Molten Globule-like State of Cytochrome C Under Conditions Simulating Those Near the Membrane Surface

Methanol-induced conformational transitions in cytochromechave been studied by near- andfar-UV circular dichroism, Trp fluorescence, microcalorimetry, and diffusion measurements. The existenceof at least two cooperative stages of transition has been shown. At the first stage, the native protein istransformed into an intermediate which has only traces of tertiary structure, but has a native-like secondarystructure content and is relatively compact; i.e., it has properties of the molten globule state. On thesecond stage, the alcohol-induced molten globule is transformed into a more helical state, typical of proteinsat high alcohol concentrations. The conditions at which the alcohol-induced molten globule exists(moderately low pH and moderately low dielectric constant) could be similar to those existing nearnegatively charged membrane surfaces. Consequently, these results might explain how the molten globulestate can be achieved under physiological conditions

Domain Coil-globule Transition in Homopolymers

The temperature-induced coil-globule transition has been studied in dilute aqueous solutions (with 200 mg/L SDS) for different fractions of poly(N-isopropylacrylamide) (PNIPAM) and poly(N-isopropylmethacrylamide) (PNIPMAM) using scanning microcalorimetry, diffusion, and size-exclusion chromatography (FPLC). It has been shown that both these polymers undergo a coil-globule transition upon temperature increase. This transition is accompanied by cooperative heat absorption and a decrease of heat capacity, which makes it similar to the cold denaturation of globular proteins. The globule-coil transition is an all-or-none process only for the fraction with the lowest molecular weight (~10x10^3), while fractions of larger molecular weights behave as if they consist of quasi-independent cooperative units, the domains . The number of domains in a macromolecule is proportional to the molecular weight of the polymer. This suggests that the domain character of cooperative transitions in large proteins does not, in principle, need evolutionary-selected amino acid sequences but can occur even in homopolymers