Ras Conformational Switching: Simulating Nucleotide-Dependent Conformational Transitions with Accelerated Molecular Dynamics

Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding Ras signaling. We characterize nucleotide-dependent conformational transition using multiple-barrier-crossing accelerated molecular dynamics (aMD) simulations. These transitions, achieved for the first time for wild-type Ras, are impossible to observe with classical molecular dynamics (cMD) simulations due to the large energetic barrier between end states. Mapping the reaction path onto a conformer plot describing the distribution of the crystallographic structures enabled identification of highly populated intermediate structures. These structures have unique switch orientations (residues 25–40 and 57–75) intermediate between GTP and GDP states, or distinct loop3 (46–49), loop7 (105–110), and α5 C-terminus (159–166) conformations distal from the nucleotide-binding site. In addition, these barrier-crossing trajectories predict novel nucleotide-dependent correlated motions, including correlations of α2 (residues 66–74) with α3-loop7 (93–110), loop2 (26–37) with loop10 (145–151), and loop3 (46–49) with α5 (152–167). The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function. In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras. Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure

Protocol for the IDEAL-2 longitudinal study: Following the experiences of people with dementia and their primary carers to understand what contributes to living well with dementia and enhances active life

Background There is a major need for longitudinal research examining the experiences of people with dementia and their primary carers, as relatively little is known about how the factors associated with capability to ‘live well’ vary over time. The main aim of the IDEAL-2 study is to investigate how and why, over time, people with dementia and their primary carers might vary in their capability to live well with dementia, whilst exploring both their use of health and care services and their unmet needs. Methods IDEAL-2 will build on the Improving the experience of Dementia and Enhancing Active Life (IDEAL) cohort of 1547 people (who, at recruitment between July 2014 and July 2016, had mild-to-moderate dementia), and their 1283 primary carers in Great Britain. The existing cohort will be enriched with additional participants with mild-to-moderate dementia (and their primary carers where available and willing) from the following groups: people with rarer forms of dementia, and/or those who are ≥90 years or < 65 years of age at time of recruitment. We will assess the primary outcome, capability to live well with dementia, and the factors influencing it using questionnaires at yearly intervals for 3 years. Additionally, we will seek to link the cohort data with administrative data to obtain information about health service use. Some participants will be invited for in-depth face-to-face interviews. The cohort study will be supplemented by linked research focusing on: the co-production of new measures of living well; including the perspectives of people with advanced dementia living in residential care settings; including people with dementia from black, Asian, and minority ethnic groups; and understanding the experience of people living with undiagnosed dementia. Discussion IDEAL-2 will provide evidence about the key indicators of, and factors associated with, living well over the course of dementia and how these differ for particular subgroups. It will tell us which combinations of services and support are most beneficial and cost-effective. Moreover, the IDEAL-2 study will gather evidence from under-researched groups of people with dementia, who are likely to have their own distinct perceptions of living well.Alzheimer’s Society & The University of Exete

The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved “arginine finger” of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras·RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling “off” to the signaling “on” state with a rate of 3 s−1. The next step is the movement of the “arginine finger” into the active site of Ras with a rate of k2 = 0.8 s−1. Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the “off” state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 = 0.1 s−1, the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the “arginine finger” movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate

Quantifying mechanisms of aeolian dust emission: field measurements at Etosha Pan, Namibia

Determining the controls on aeolian dust emissions from major sources is necessary for reliable quantification of atmospheric aerosol concentrations and fluxes. However, ground-based measurements of dust emissions at-source are rare and of generally short duration, failing to capture the annual cycle. Here, we provide new insights into dust dynamics by measuring aerosol concentrations and meteorological conditions for a full year (July 2015-June 2016) at Etosha Pan, Namibia, a globally significant dust source. Surface deployed field instrumentation provided 10-minute averaged data on meteorological conditions, aerosol concentration (mg/m3), and horizontal dust flux (g/m2/min10). A Doppler LiDAR provided additional data for some of the period. 51 significant dust events were identified in response to strong E-ENE winds. We demonstrate that these events occurred throughout the year and were not restricted to the austral winter, as previously indicated by satellite observations. Peak horizontal flux occurred in the spring (November) due to strengthening erosive winds and highly desiccating conditions increasing surface erodibility. We identify a strong seasonal differentiation in the meteorological mechanisms controlling dust uplift; low-level jets (LLJ) on dry winter mornings (61% of all events), and cold pool outflows (CPO) in humid summer evenings (39% of events). Significantly, we demonstrate a very strong bias towards the contribution of low frequency and high magnitude events, with nearly 31% of annual horizontal dust flux generated by only 6 individual events. Our study demonstrates how longer-term (≈1 year), ground-based, and at-source field measurements can radically improve interpretations of dust event dynamics and controls at major source locations