Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques

A protein dynamically
samples multiple conformations, and the conformational
dynamics enables protein function. Most biophysical measurements are
ensemble-based, with the observables averaged over all members of
the ensemble. Though attainable, the decomposition of the observables
to the constituent conformational states can be computationally expensive
and ambiguous. Here we show that the incorporation of single-molecule
fluorescence resonance energy transfer (smFRET) data resolves the
ambiguity and affords protein ensemble structures that are more precise
and accurate. Using K63-linked diubiquitin, we characterize the dynamic
domain arrangements of the model system, with the use of chemical
cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray
scattering (SAXS), and smFRET techniques. CXMS allows the modeling
of protein conformational states that are alternatives to the crystal
structure. SAXS provides ensemble-averaged low-resolution shape information.
Importantly, smFRET affords state-specific populations, and the FRET
distances validate the ensemble structures obtained by refining against
CXMS and SAXS restraints. Together, the integrative use of bulk and
single-molecule techniques affords better insight into protein dynamics
and shall be widely implemented in structural biology.