Temperature-dependent hydrogen deuterium exchange shows impact of analog binding on adenosine deaminase flexibility but not embedded thermal networks.

The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 106-fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis.

Accidental hypothermia: direct evidence for consciousness as a marker of cardiac arrest risk in the acute assessment of cold patients.

BACKGROUND: Rapid stratification of the risk of cardiac arrest is essential in the assessment of patients with isolated accidental hypothermia. Traditional methods based on measurement of core temperature are unreliable in the field. Behavioural observations have been used as predictors of core temperature and thus indirect predictors of cardiac arrest. This study aims to quantify the direct relationship between observed conscious level and cardiac arrest. METHODS: Retrospective case report analysis identified 114 cases of isolated accidental hypothermia meeting inclusion criteria. Level of consciousness in the acute assessment and management phase was classified using the AVPU system with an additional category of "Alert with confusion"; statistical analysis then related level of consciousness to incidence of cardiac arrest. RESULTS: All patients who subsequently suffered cardiac arrest showed some impairment of consciousness (p < < .0001), and the risk of arrest increased directly with the level of impairment; none of the 33 fully alert patients arrested. In the lowest impairment category, Alert confused, a quarter of the 12 patients went on to arrest, while in the highest Unresponsive category, two thirds of the 43 patients arrested. Where core temperature was available (62 cases), prediction of arrest by consciousness level was at least as good as prediction from core temperature. CONCLUSIONS: This study provides retrospective analytical evidence that consciousness level is a valid predictor of cardiac arrest risk in isolated accidental hypothermia; the importance of including confusion as a criterion is a new finding. This study suggests the use of consciousness alone may be at least as good as core temperature in cardiac arrest risk prediction. These results are likely to be of particular relevance to the management of accidental hypothermia in the pre-hospital and mass casualty environment, allowing for rapid and accurate triage of hypothermic patients.

Hydrogen-Deuterium Exchange within Adenosine Deaminase, a TIM Barrel Hydrolase, Identifies Networks for Thermal Activation of Catalysis.

Proteins are intrinsically flexible macromolecules that undergo internal motions with time scales spanning femtoseconds to milliseconds. These fluctuations are implicated in the optimization of reaction barriers for enzyme catalyzed reactions. Time, temperature, and mutation dependent hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been previously employed to identify spatially resolved, catalysis-linked dynamical regions of enzymes. We now extend this technique to pursue the correlation of protein flexibility and chemical reactivity within the diverse and widespread TIM barrel proteins, targeting murine adenosine deaminase (mADA) that catalyzes the irreversible deamination of adenosine to inosine and ammonia. Following a structure-function analysis of rate and activation energy for a series of mutations at a second sphere phenylalanine positioned in proximity to the bound substrate, the catalytically impaired Phe61Ala with an elevated activation energy (Ea = 7.5 kcal/mol) and the wild type (WT) mADA (Ea = 5.0 kcal/mol) were selected for HDX-MS experiments. The rate constants and activation energies of HDX for peptide segments are quantified and used to assess mutation-dependent changes in local and distal motions. Analyses reveal that approximately 50% of the protein sequence of Phe61Ala displays significant changes in the temperature dependence of HDX behaviors, with the dominant change being an increase in protein flexibility. Utilizing Phe61Ile, which displays the same activation energy for kcat as WT, as a control, we were able to further refine the HDX analysis, highlighting the regions of mADA that are altered in a functionally relevant manner. A map is constructed that illustrates the regions of protein that are proposed to be essential for the thermal optimization of active site configurations that dominate reaction barrier crossings in the native enzyme.