SEQUENTIAL ASSEMBLY OF COLLAGEN REVEALED BY ATOMIC-FORCE MICROSCOPY

Most polymers which comprise biological filaments assemble by two mech anisms: nucleation and elongation or a sequential, stepwise process in volving a hierarchy of intermediate species. We report the application of atomic force microscopy (AFM) to the study of the early events in the sequential or stepwise mode of assembly of a macromolecular filame nt. Collagen monomers were assembled in vitro and the early structural intermediates of the assembly process were examined by AFM and correl ated with turbidimetric alterations in the assembly mixture. The assem bly of collagen involved a sequence of distinctive filamentous species which increased in both diameter and length over the time course of a ssembly. The first discrete population of collagen oligomers were 1-2 nm in diameter (300-500 nm in length); at later time points, filaments similar to 2-6 nm in diameter (>10 mu m in length) many with a conspi cuous similar to 67-nm axial period were observed. Occasional mature c ollagen fibrils with a similar to 67-nm axial repeat were found late i n the course of assembly. Our results are consistent with initial end- to-end axial association of monomers to form oligomers followed by lat eral association into higher-order filaments. On this basis, there app ears to be at least two distinctive types of structural interactions ( axial and lateral) which are operative at different levels in the asse mbly hierarchy of collagen.