4.5 Backbone Dynamics of L-hIGFBP2 from 15N
relaxation
The experiments for dynamics studies of 15N-labeled
hL-IGFBP2 in the presence and absence of IGF-1 were performed at 20 °C
on a Bruker 800 MHz NMR spectrometer equipped with a cryogenic probe.
For R1 measurements, eight different time points were
collected with relaxation delay periods of 0.01 s, 0.05 s, 0.1 s, 0.2 s,
0.4 s, 0.8 s, 1 s, and 1.5 s. R2 and R1ρmeasurements were carried out at eight different time points with
relaxation delay periods of 0.01 s, 0.03 s, 0.05 s, 0.1 s, 0.13 s, 0.17
s, 0.2 s, 0.23 s. For the R1, R2,
R1ρ experiments: 2 scans with an interscan relaxation
delay of 2.5 s, 256 points, and a spectral window of 24 ppm (65 ms
acquisition time) were used in the 15N dimension. The1H dimension was acquired using 2048 points
(t max = 106 ms) over a 12-ppm spectral width. For
R1ρ a 4 ms spin-lock block with a field strength of 2
kHz was used. The same data sets with identical time points, relaxation
delays, and experimental time were recorded for the IGF-1-bound form of
L-h IGFBP2. All the relaxation experiments were processed with a
shifted sine-bell function and zero-filled with twice the time domain
points in both dimensions.
[15N-1H] Het-NOE spectra for the
free and bound form of L-h IGFBP2 were recorded with 32 transients
for 23 h each with 2048 and 256 points in the direct and indirect
dimensions, respectively. An interscan delay of 5 s was used and
acquisition times were 80 ms in the direct and 65 ms in the indirect
dimension.
An approximate S2 to estimate the conformational
entropy of the NH bond vector was calculated using Equation 1 and the
entropy was calculated using Equation 2.