Microsecond timescale backbone conformational dynamics in ubiquitin studied with NMR R1rho relaxation experiments

Abstract

NMR spin relaxation experiments are used to characterize the dynamics of the backbone of ubiquitin. Chemical exchange processes affecting residues Ile 23, Asn 25, Thr 55, and Val 70 are characterized using on- and off-resonance rotating-frame 15N R1rho relaxation experiments to have a kinetic exchange rate constant of 25,000 sec(-1) at 280 K. The exchange process affecting residues 23, 25, and 55 appears to result from disruption of N-cap hydrogen bonds of the alpha-helix and possibly from repacking of the side chain of Ile 23. Chemical exchange processes affecting other residues on the surface of ubiquitin are identified using 1H-15N multiple quantum relaxation experiments. These residues are located near or at the regions known to interact with various enzymes of the ubiquitin-dependent protein degradation pathway.

Figure 1.

Conformational exchange contribution to transverse relaxation, R_ex, for ubiquitin at T = 298 K (open circles) and T = 280 K (filled circles) at B₀ = 11.7 T.

Figure 2.

Conformational exchange contribution to transverse relaxation, R_ex as a function of the effective field, ω_e, at a static magnetic field of 11.7 T and T = 298 K for Ile 23 (circles) and Asn 25 (squares) in ubiquitin.

Figure 3.

Cartoon representation of ubiquitin that highlights the positions of the exchanging residues Ile 23, Asn 25, Thr 55, Val 70 (A). Regions characterized by different secondary structure are represented with different colors: helices ,red; β-strands, yellow; coil and turns, blue. The boxed region is shown as an expansion in B. Each residue is labeled at the backbone N atom. The dashed lines indicate hydrogen bonds.

Figure 4.

Conformational exchange contribution to transverse relaxation, R_ex, as a function of the effective field, ω_e at T = 280 K. Circles and squares represent data collected at a static magnetic field of 11.7 T and 14.1 T, respectively. Lines are the results of simultaneously fitting the data at 11.7 T (circles) and 14.1 T (dashed), using equation 2.

Figure 5.

Amide ¹H-¹⁵N differential relaxation rate constants for ZQ and DQ coherences, ΔR_MQ, for ubiquitin measured at 11.7 T and T = 280 K.

Figure 6.

Dependence of the ¹⁵N chemical shift on side chain χ₁ dihedral angle. ¹⁵N secondary chemical shifts are plotted as a function of side chain χ₁ torsional angle for all isoleucine (A) and asparagine (B) residues in the RefDB chemical shift database (Zhang et al. 2003).

Figure 7.

Amide ¹H-¹⁵N ΔR_MQ for ubiquitin measured at 11.7 T and T = 280 K. Data are represented with different colors on the surface of the protein. Residues with ΔR_MQ < 3 are represented as gray, residues with 3 ≤ ΔR_MQ < 5 are yellow, residues with 5 ≤ ΔR_MQ < 8 are orange, residues with 8 ≤ ΔR_MQ < 15 are red, and residues with ΔR_MQ ≥ 15 are purple. The opaque residues are those known to be directly involved in the interaction with E1 and E2 enzymes. All other residues are depicted as transparent.