The effects of volume exclusion and viscosity on collagen fiber nucleation and network morphology

Protein behavior, including protein-protein association and self-assembly, differs in dilute buffers compared to in the crowded environments that are typical in vivo. We performed a comprehensive study of the impacts of crowding on collagen self-assembly into fibers using a range of polysaccharides and polyethylene glycols (PEG) at volume fractions in the physiological range as well as small molecule analogues of these species. We find that crowding accelerates collagen fiber nucleation in proportion to excluded volume without changes to collagen secondary structure or thermal stability, with the acceleration greater for PEGs than for polysaccharides at the same volume fraction. Additionally, we observed the competing effects of volume fraction and viscosity on nucleation, with volume fraction dominant in setting the collagen fiber nucleation time at high degrees of crowding. We show, consistent with previous findings on collagen gelation as a function of temperature, that fiber and network morphological and mechanical differences in collagen gels formed from these solutions is determined primarily by changes in nucleation time, with short nucleation times leading to softer collagen networks with abundant, thin fibers and long nucleation times resulting in stiffer networks with sparse, thick fiber bundles. STATEMENT OF SIGNIFICANCE: We studied the effects of several macromolecules and small molecule analogues on collagen self-assembly and the resulting fiber and network properties of collagen gels. We observed competing effects of excluded volume and viscosity on collagen nucleation depending on the volume fraction of the co-solute and assert that the morphological changes in the resulting gels are due to changes to nucleation time alone. This work elucidates the various effects of co-solutes on collagen self-assembly and is of use for control of collagen material properties and for understanding crowding and related effects on other self-associating proteins.