Excluded Volume Effects in Molecular Biology and Extracellular Matrix Biochemistry: Biophysical Considerations and Molecular Modeling

Abstract

Macromolecular crowding leads to the excluded volume effect (EVE) and governs biological processes at intra- and extracellular level. We demonstrate that macromolecular size, charge and charge density together determine EVE with powerful consequences. Crowding applied to a well defined simple system, reverse-transcriptase-polymerase chain reaction, improved sensitivity by ~10-fold, specificity, efficiency and product yield by ~2-fold with enhanced thermal stability and processivity of DNA polymerase. As major reason for this improvement we identified an increase of melting temperatures of nucleic acid hybrids by 5-8B0C under crowding, as also corroborated by molecular modeling showing structural stabilization of nucleic acid duplexes via intact H-bonds upon heating. Crowding a more complex system, fibroblast cultures, enhanced enzymatically controlled collagen deposition by 10-fold. In correlation with their hydrodynamic radii, anionic macromolecules with greater surface charge density lead to greater collagen deposition. Thus we demonstrate the huge potential of reverse bioengineering crowded conditions for in vitro applications.