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3
APPLICATION NOTE
To validate the MST results, experiments using other
biophysical methods including isothermal titration
calorimetry (ITC), surface plasmon resonance (SPR)
and bio-layer interferometry (BLI) were performed
Using ITC, we were able to confirm that RTA directly
interacts with P1-P2 heterodimer. However, we were
not able to measure the K
d
for the mutant proteins due
to technical limitations of the method. BLI showed that
mutations of individual dimer components as well as
assembly of functional binding partner as homodimer
results in weakening of affinity of the interaction based
on BLI results. On the other hand, SPR allowed us to
visually see the differences in binding potency of various
assemblies of the functional dimer. However, the
quantification of affinities of these binding events was
hindered due lack of clear dissociation state (Table 1).
Figure 1: Characterization of human ribosomal stalk P1-P2 complexes. (A)
Alignment of C-terminal amino acid sequences of human ribosomal P1, P2
protein. The arrow and box indicate the deletion site from the C-termini (ΔC). (B)
Models of P1-P2 and P2-P2 complexes with deleted in the conserved C-terminal
16 amino acids sequences. (C) Size exclusion chromatography (SEC) of purified
protein complexes. Purified complexes (10 μg of protein) were analyzed by
SDS-PAGE (upper panel) and circular dichroism (CD) (lower panel). (D) Molecular
masses of protein species detected by native mass spectrometry. *Calculated
molecular masses (MMcal) compared with molecular masses determined by
native-MS (MMmeasured).
Protein complex MMcal (Da)*
MMmeasured
(Da)*
P1-met 12010.6 12009.98±06
P1ΔC-met 10357.8 10357.52±0.57
P2 12292.7 12292.66±0.10
P2ΔC-met 10357.8 10357.68±0.10
P1-met-P2 24302.6 24302.45±7.14
P1-met-P2ΔC-met 22368.4 22367.97±2.54
P1ΔC-met-P2 22499.5 22497.71±5.11
P1ΔC-met-P2ΔC-met 20564.6 20563.95±11.51
P2-P2 24585.4 24586.09±8.81
P2ΔC-met-P2ΔC-me 20715.7 20727.95±11.10
A
B
C
D
