Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Solution Scattering 155
structure. They further show that TnI undergoes a compaction upon addition of
Ca 2+ . The measurements of the radius of gyration for TnI in the ternary troponin
complex indicate a more compact structure than was observed for the binary
complex, suggesting that of TnT influences the TnC–TnI interaction.
The TnC–TnI interaction has proven very difficult to study, and there is a
growing amount of apparently contradictory data using different experimental
approaches. One difficulty the system has for study using scattering techniques
is that there are no high-resolution structural data on TnI and its structure is
very unusual. TnI is not a “typical” globular protein, and therefore cannot be
modeled simply as an ellipsoid to aid in the interpretation of scattering data.
Based on the neutron data for the binary complex, a model was developed (27)
in which TnI forms a superhelical structure around the 4Ca 2+ /TnC extending
into an incomplete donut shaped structures that project beyond the TnC at each
end. The diameter of the TnI central spiral is 12 Å, close to that expected for an
α helix, and it passes through or near the two hydrophobic clefts in each globular
domain of 4Ca 2+ /TnC. Based on this model, it was proposed that the C-terminal
domain of TnC anchors TnI while the N-terminal domain alternately binds
and releases TnI in response to the Ca 2+ signal. Recent data from NMR,
crosslinking FTIR, and crystallography have yielded a high-resolution model
for Tn(–TnI) that is based on the earlier neutron model (28). Thus, although
there remains much work to be done before we fully understand the complex
interactions within the troponin complex, the neutron scattering experiments
on TnC–TnI (25) and troponin (26) have yielded some important consensus
conclusions. In the presence of the intact TnI, TnC has a fully extended structure
that is very similar to its crystal structure. Further, TnI has an even more
elongated structure, although the details of that structure show a dependence
on TnT and Ca 2+ .
4. Notes
1. Protein aggregation and the importance of accurate protein concentration determinations
and I0 calibration: Nonspecific protein aggregation can be fatal in a
small-angle scattering experiment. Analysis of scattering data from solutions containing
aggregates will give structural parameters that are systematically larger
than the correct values. Because the scattering signal is proportional to the square
of the molecular weight of the scattering particle, even small amounts of aggregated
material in a solution will bias the data very severely toward the aggregated
species. If it is severe enough, aggregation can be seen in the very small angle
scattering data as a deviation from linearity in the Guinier plots (see Subheading
3.5.1.). However, samples with small amounts of aggregation can yield perfectly
linear Guinier plots. Another signature of aggregation can be seen in the concentration
dependence of the scattering data. For a homogeneous, monodisperse
solution of protein molecules, the shape of the scattering profile should be either