Erin M. Poss

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

Defining specific pathways for efficient heat transfer from protein-solvent interfaces to their active sites represents one of the compelling and timely challenges in our quest for a physical description of the origins of enzyme catalysis. Enzymatic hydrogen tunneling reactions constitute excellent systems in which to validate experimental approaches to this important question, given the inherent temperature independence of quantum mechanical wave function overlap. Herein, we present the application of hydrogen-deuterium exchange coupled to mass spectrometry toward the spatial resolution of protein motions that can be related to an enzyme's catalytic parameters. Employing the proton-coupled electron transfer reaction of soybean lipoxygenase as proof of principle, we first corroborate the impact of active site mutations on increased local flexibility and, second, uncover a solvent-exposed loop, 15-34 Å from the reactive ferric center whose temperature-dependent motions are demonstrated to mirror the enthalpic barrier for catalytic C-H bond cleavage. A network that connects this surface loop to the active site is structurally identified and supported by changes in kinetic parameters that result from site-specific mutations.

Key points Several brain regions are thought to sense changes in tissue CO 2 /H + to regulate breathing (i.e. central chemoreceptors) including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). Mechanism(s) underlying RTN chemoreception involve direct activation of RTN neurons by H + ‐mediated inhibition of a resting K + conductance and indirect activation of RTN neurons by purinergic signalling, most likely from CO 2 /H + ‐sensitive astrocytes. Here, we confirm that activation of P2 receptors in the RTN stimulates cardiorespiratory activity, and we show at the cellular and systems level that purinergic signalling is not essential for CO 2 /H + sensing in the NTS or medullary raphe. These results support the possibility that purinergic signalling is a unique feature of RTN chemoreception. Abstract Several brain regions are thought to function as important sites of chemoreception including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). In the RTN, mechanisms of chemoreception involve direct H + ‐mediated activation of chemosensitive neurons and indirect modulation of chemosensitive neurons by purinergic signalling. Evidence suggests that RTN astrocytes are the source of CO 2 ‐evoked ATP release. However, it is not clear whether purinergic signalling also influences CO 2 /H + responsiveness of other putative chemoreceptors. The goals of this study are to determine if CO 2 /H + ‐sensitive neurons in the NTS and medullary raphe respond to ATP, and whether purinergic signalling in these regions influences CO 2 responsiveness in vitro and in vivo . In brain slices, cell‐attached recordings of membrane potential show that CO 2 /H + ‐sensitive NTS neurons are activated by focal ATP application; however, purinergic P2‐receptor blockade did not affect their CO 2 /H + responsiveness. CO 2 /H + ‐sensitive raphe neurons were unaffected by ATP or P2‐receptor blockade. In vivo , ATP injection into the NTS increased cardiorespiratory activity; however, injection of a P2‐receptor blocker into this region had no effect on baseline breathing or CO 2 /H + responsiveness. Injections of ATP or a P2‐receptor blocker into the medullary raphe had no effect on cardiorespiratory activity or the chemoreflex. As a positive control we confirmed that ATP injection into the RTN increased breathing and blood pressure by a P2‐receptor‐dependent mechanism. These results suggest that purinergic signalling is a unique feature of RTN chemoreception.

ABSTRACT Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

The purpose of this investigation was to determine whether people could accurately perceive physiological foot temperature during brief bouts of treadmill running in different combinations of shoe and sock models, and also how perception of comfort was influenced. Sixteen young adult males (21.3  0.8 years, 181.8  1 cm, 74.6  1.5 kg) participated in two separate studies where they alternated running and resting for 10 min each with temperature probes attached at two sites on the lateral dorsal aspect of the right foot. Subjects reported perceptions of foot comfort and temperature after each run using 10 cm visual analogue scales. In the first experiment, different sock models were tested with the same shoe model; in the second experiment, different shoe models were tested with the same sock model. Foot temperature did not differ statistically as a function of shoe or sock model in either experiment. Subjects did not perceive any difference in foot temperature in the shoe experiment, but perceived their foot as being cooler when wearing either a polyester sock or a calf compression sleeve and more comfortable when wearing shoes with less mass. Taken together, the results suggest that subjects’ perceptions of foot temperature may not coincide with physiological foot temperature and are more strongly influenced by sock characteristics than shoe characteristics. Further, shoe mass (but not sock fiber weave or composition) may impact comfort perception by subjects.