  DNA in sperm cells must undergo an extreme compaction to almost crystalline
packing levels. To produce this dense packing, DNA is condensed by protamine, a
positively charged protein that loops the DNA into a toroid. Our goal is to
determine the pathway and mechanism for toroid formation. We first imaged
short-length (L=217-1023 nm) DNA molecules in 0-5.0 $\mu$M protamine using an
atomic force microscope (AFM). At low protamine concentrations (0.2-0.6
$\mu$M), molecules dramatically condensed, folding into a flower structure.
Dynamic folding measurements of the DNA using a tethered particle motion (TPM)
assay revealed a corresponding, initial folding event, which was >3 loops at
L=398 nm. The initial folding event was made up of smaller (<1 loop) events
that had similar dynamics as protamine-induced bending. This suggests that
flowers form in an initial step as protamine binds and bends the DNA. It was
not until higher protamine concentrations (>2 $\mu$M) that DNA in the AFM assay
formed small (<10 loop), vertically packed toroids. Taken together, these
results lead us to propose a nucleation-growth model of toroid formation that
includes a flower intermediate. This pathway is important in both in vivo DNA
condensation and in vitro engineering of DNA nanostructures.
