Anti-Ubiquitin polyclonal antibody

Pulsed temperature-jump (T-jump) spectroscopy with infrared (IR) detection has been widely used to study biophysical processes occurring from nanoseconds to similar to 1 ms with structural sensitivity. However, many systems exhibit structural dynamics on time scales longer than the millisecond barrier that is set by the time scale for thermal relaxation of the sample. We developed a linear and nonlinear infrared spectrometer coupled to an intensity-modulated continuous wave (CW) laser to probe T-jump-initiated chemical reactions from <1 ms to seconds. Time-dependent modulation of the CW laser leads to a <1 ms heating time as well as a constant final temperature (+/- 3%) for the duration of the heating time. Temperature changes of up to 75 degrees C in D2O are demonstrated, allowing for nonequilibrium measurements inaccessible to standard pulsed optical T-jump setups. T-jump linear absorption, pump-probe, and two-dimensional IR (2D IR) spectroscopy are applied to the unfolding and refolding of ubiquitin and a model intercalated motif (i-motif) DNA sequence, and analysis of the observed signals is used to demonstrate the limits and utility of each method. Overall, the ability to probe temperature-induced chemical processes from <1 ms to many seconds with 2D IR spectroscopy provides multiple new avenues for time-dependent spectroscopy in chemistry and biophysics.

At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover. To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers auto-inhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.