The Carboxyl-Terminal Segment of Apolipoprotein A-V Undergoes a Lipid-Induced Conformational Change

Apolipoprotein (apo) A-V is a 343-residue, multidomain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetraproline sequence (Pro293-Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48-residue segment beyond the tetraproline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high-density lipoprotein with Stokes’ diameters\u3e17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far-UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant R-helical secondary structure in the presence of the lipid mimetic solvent trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly 15N- and 15N/13C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOSþ reveal the peptide adopts R-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic R-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V

NMR study of the role of M42 in the solution dynamics of E. COLI dihydrofolate reductase

It is widely recognized that key positions throughout a protein’s structure contribute unequally to function. In light of recent studies that suggest protein dynamics are required for function, a number of these residues may serve to promote motions required for ligand binding and catalysis. In the present NMR study, the conformational dynamics of the dihydrofolate reductase (DHFR) mutant M42W, in the presence of methotrexate and NADPH, are characterized and compared to the wild-type enzyme. M42 is distal to the active site, yet the M42W substitution regulates catalysis and ligand affinity, and is therefore analogous to an allosteric modulator of DHFR function. To gain understanding of how this mutation regulates activity, we employ a “pandynamic” strategy by measuring conformational fluctuations of backbone amide and side-chain methyl groups on multiple timescales. Changes in ps-ns dynamics indicate that the mutational effects are propagated throughout a network of interacting residues within DHFR, consistent with a role for M42 as a dynamic communication hub. On the μs-ms timescale, mutation increases the rate of switching in the catalytic core. Mutation also introduces switching in the adenosine binding subdomain that occurs at a higher frequency than in the catalytic core, and which correlates with the rate of product release for M42W-DHFR. Finally, a structurally inferred analysis of side-chain dynamics suggests that the M42W mutation dampens motional contributions from non-local effects. These data show that the M42W mutation alters the dynamics of DHFR and are consistent with theoretical analysis that suggests the mutation disrupts motion that promotes catalysis

Royal Coachman (41CM111) An Early Middle Archaic Site along Cordova Creek in Comal County, Texas

The Royal Coachman site, 41CM111, is bisected by FM 306 near the highway’s eastern crossing of the Guadalupe River. Archeologists from the Texas Department of Transportation conducted extensive excavations at the site in 1980 and the Center for Archaeological Research carried out fieldwork related to geomorphic assessment of the deposits in 2002. The site contains at least three archeological components, an upper zone that may be of late Middle Archaic age and two commingled lower zones that are early Middle Archaic in age and contain a mix of Nolan/ Pandale and Bell-Andice/Early Triangular points. The deeper, more strongly manifested archeological components are associated with a dense burned rock sheet midden composed of two in situ features among a dense scatter of highly disturbed burned rock. Radiocarbon assays place this zone at between 5320–5880 years BP. The lithic assemblage is suggestive of tool manufacture and retooling activities and the projectile point collection is dominated by Early Triangular points and preforms. It is the recommendation of CAR that site 41CM111 warrants designation as a State Archeological Landmark and is eligible for listing on the National Register of Historic Places because of its significant contribution to a better understanding of the regional archeological record. We believe that these investigations have resulted in an appropriate level of work to evaluate the archeological property within the highway ROW and no further work is currently recommended. We also recommend that TxDOT consider avoidance as an option during future construction activities to avert impact to remaining deposits. In addition, if new ROW is acquired, additional work is warranted to determine the extent and significance of currently unidentified material. This work was conducted under Texas Antiquities Committee Permit No. 2807

Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State

The arduous task of rationally designing small molecule enzyme inhibitors is complicated by the inherent flexibility of the protein scaffold. To gain insight into the changes in dynamics associated with small molecule based inhibition, we have characterized, using NMR spectroscopy, E. coli dihydrofolate reductase in complex with two drugs: methotrexate and trimethoprim. The complexes allowed the intrinsic dynamic effects of drug binding to be revealed within the context of the “closed” structural ensemble. Binding of both drugs results in an identical decoupling of global motion on the micro- to millisecond timescale. Consistent with a change in overall dynamic character, the drugs’ perturbations to pico- to nanosecond backbone and side-chain methyl dynamics are also highly similar. These data show that the inhibitors simultaneously modulate slow concerted switching and fast motions at distal regions of DHFR, providing a dynamic link between the substrate binding site and distal loop residues known to affect catalysis

Evidence for Diverse Biogeochemical Drivers of Boreal Forest New Particle Formation

New particle formation (NPF) is an important contributor to particle number in many locations, but the chemical drivers for this process are not well understood. Daytime NPF events occur regularly in the springtime Finnish boreal forest and strongly impact aerosol abundance. In April 2014 size-resolved chemical measurements of ambient nanoparticles were made using the Time-of-Flight Thermal Desorption Chemical ionization Mass Spectrometer and we report results from two NPF events. While growth overall was dominated by terpene oxidation products, newly formed 20-70nm particles showed enhancement in apparent alkanoic acids. The events occurred on days with rapid transport of marine air, which correlated with low background aerosol loading and higher gas phase methanesulfonic acid levels. These results are broadly consistent with previous studies on Nordic NPF but indicate that further attention should be given to the sources and role of non-terpenoid organics and the possible contribution of transported marine compounds in this process. Plain Language Summary Clouds are an enormously important part of the climate system because they control the radiation entering and leaving the Earth. Clouds form as water condenses onto small particles called cloud condensation nuclei. These particles can be directly emitted from the Earth's surface, like sea spray, for example, or they can form in the atmosphere out of precursor gases. We have measured the composition of these atmosphere-formed particles to understand better how this process works in the Nordic boreal forest. We found that a diverse mix of processes and molecules are likely involved, possibly including the transport of materials from the ocean. While these results will ultimately lead to a better understanding of ocean-land-cloud interactions, they currently indicate that more work is needed to learn the processes involved.Peer reviewe

Low Serum Hepcidin Is Associated With Reduced Shortâ Term Survival in Adults With Acute Liver Failure

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149282/1/hep30486_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149282/2/hep30486.pd

Addressing Systemic Factors Related to Racial and Ethnic Disparities among Older Adults in Long-Term Care Facilities

Disparities in older adults’ care and experiences in long-term care facilities (LTCFs) such as nursing homes and assisted living/residential care communities reflect disparities in the broader society. Various policies and institutional practices related to economic opportunity, education, housing, health care, and retirement financing have created and maintain inequitable social structures in the United States. This chapter describes racial and ethnic disparities among older adults in LTCFs in the United States and the systemic factors associated with those disparities. It presents a conceptual framework for understanding the role of structural racism in the racial and ethnic inequities experienced by LTCF residents. In the framework, structural racism directly contributes to racial and ethnic inequities among LTCF residents through LTCF-related policies and practices. Structural racism also indirectly causes disparities among LTCF residents through health and economic disparities. The chapter describes current efforts that address the effects of structural racism within LTCFs and concludes with practice and policy recommendations to redress racial and ethnic disparities among LTCF residents

Laboratory study on new particle formation from the reaction OH + SO2: influence of experimental conditions, H2O vapour, NH3 and the amine tert-butylamine on the overall process

Peer reviewe

Steady state free radical budgets and ozone photochemistry during TOPSE

A steady state model, constrained by a number of measured quantities, was used to derive peroxy radical levels for the conditions of the Tropospheric Ozone Production about the Spring Equinox (TOPSE) campaign. The analysis is made using data collected aboard the NCAR/NSF C-130 aircraft from February through May 2000 at latitudes from 40° to 85°N, and at altitudes from the surface to 7.6 km. HO2 + RO2 radical concentrations were measured during the experiment, which are compared with model results over the domain of the study showing good agreement on the average. Average measurement/model ratios are 1.04 (σ = 0.73) and 0.96 (σ = 0.52) for the MLB and HLB, respectively. Budgets of total peroxy radical levels as well as of individual free radical members were constructed, which reveal interesting differences compared to studies at lower latitudes. The midlatitude part of the study region is a significant net source of ozone, while the high latitudes constitute a small net sink leading to the hypothesis that transport from the middle latitudes can explain the observed increase in ozone in the high latitudes. Radical reservoir species concentrations are modeled and compared with the observations. For most conditions, the model does a good job of reproducing the formaldehyde observations, but the peroxide observations are significantly less than steady state for this study. Photostationary state (PSS) derived total peroxy radical levels and NO/NO2ratios are compared with the measurements and the model; PSS-derived results are higher than observations or the steady state model at low NO concentrations

Structure and Dynamics of the G121V Dihydrofolate Reductase Mutant: Lessons from a Transition-State Inhibitor Complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on 15N and 2H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect