Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used a new technique of site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA - an essential chromosomal gene of E. coli encoding dihydrofolate reductase (DHFR) - to determine how changes in protein stability, activity and abundance affect fitness. In total, 27 E.coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance; suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30oC. At 42oC, on the other hand, the picture was mixed, yet remarkable: a few strains carrying mutant DHFR proteins aggregated rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42oC by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation

Efficiency of the EmERGE Pathway of Care in Five European HIV Centres

Objective: We aimed to calculate the efficiency of the EmERGE Pathway of Care in five European HIV clinics, developed and implemented for medically stable people living with HIV. Methods: Participants were followed up for 1 year before and after implementation of EmERGE, between April 2016 and October 2019. Micro-costing studies were performed in the outpatient services of the clinics. Unit costs for outpatient services were calculated in national currencies and converted to US$2018 OECD purchasing parity prices to enable between clinic comparisons in terms of outcomes and costs. Unit costs were linked to the mean use of services for medically stable people living with HIV, before and after implementation of EmERGE. Primary outcome measures were CD4 count and viral load; secondary outcomes were patient activation (PAM13) and quality of life (PROQOL-HIV). Out-of-pocket expenditure data were collected. Results: There were 2251 participants: 87-93$ purchasing parity prices. Primary and secondary outcome measures of participants did not change during the study. Conclusions: EmERGE is acceptable and provided cost savings in different socio-economic settings. Antiretroviral drug costs remain the main cost drivers in medically stable people living with HIV. While antiretroviral drug prices in local currencies did not differ that much between countries, conversion to US$ purchasing parity prices revealed antiretroviral drugs were more expensive in the least wealthy countries. This needs to be taken into consideration when countries negotiate drug prices with pharmaceutical vendors. Greater efficiencies can be anticipated by extending the use of the EmERGE Pathway to people with complex HIV infection or other chronic diseases. Extending such use should be systematically monitored, implementation should be evaluated and funding should be provided to monitor and evaluate future changes in service provision.info:eu-repo/semantics/publishedVersio

Discovering Conformational Sub-States Relevant to Protein Function

Background: Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these substates present significant challenges for their identification and characterization. Methods and Findings: To overcome these challenges we have developed a new computational technique, quasianharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions: Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. © 2011 Ramanathan et al

Coupled Motions Direct Electrons along Human Microsomal P450 Chains

Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes

Evolutionarily Conserved Linkage between Enzyme Fold, Flexibility, and Catalysis

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design

A Rigidifying Salt-Bridge Favors the Activity of Thermophilic Enzyme at High Temperatures at the Expense of Low-Temperature Activity

Although enzymes from thermophiles thriving in hot habitats are more stable than their mesophilic homologs, they are often less active at low temperatures. One theory suggests that extra stabilizing interactions found in thermophilic enzymes may increase their rigidity and decrease enzymatic activity at lower temperatures. We used acylphosphatase as a model to study how flexibility affects enzymatic activity. This enzyme has a unique structural feature in that an invariant arginine residue, which takes part in catalysis, is restrained by a salt-bridge in the thermophilic homologs but not in its mesophilic homologs. Here, we demonstrate the trade-offs between flexibility and enzymatic activity by disrupting the salt-bridge in a thermophilic acylphosphatase and introducing it in the mesophilic human homolog. Our results suggest that the salt-bridge is a structural adaptation for thermophilic acylphosphatases as it entropically favors enzymatic activity at high temperatures by restricting the flexibility of the active-site residue. However, at low temperatures the salt-bridge reduces the enzymatic activity because of a steeper temperature-dependency of activity

The Alkaline Hydrolysis of Sulfonate Esters: Challenges in Interpreting Experimental and Theoretical Data

Sulfonate ester hydrolysis has been the subject of recent debate, with experimental evidence interpreted in terms of both stepwise and concerted mechanisms. In particular, a recent study of the alkaline hydrolysis of a series of benzene arylsulfonates (Babtie et al., Org. Biomol. Chem. 10, 2012, 8095) presented a nonlinear Brønsted plot, which was explained in terms of a change from a stepwise mechanism involving a pentavalent intermediate for poorer leaving groups to a fully concerted mechanism for good leaving groups and supported by a theoretical study. In the present work, we have performed a detailed computational study of the hydrolysis of these compounds and find no computational evidence for a thermodynamically stable intermediate for any of these compounds. Additionally, we have extended the experimental data to include pyridine-3-yl benzene sulfonate and its N-oxide and N-methylpyridinium derivatives. Inclusion of these compounds converts the Brønsted plot to a moderately scattered but linear correlation and gives a very good Hammett correlation. These data suggest a concerted pathway for this reaction that proceeds via an early transition state with little bond cleavage to the leaving group, highlighting the care that needs to be taken with the interpretation of experimental and especially theoretical data

Transcriptome Analysis of the Hippocampal CA1 Pyramidal Cell Region after Kainic Acid-Induced Status Epilepticus in Juvenile Rats

Molecular mechanisms involved in epileptogenesis in the developing brain remain poorly understood. The gene array approach could reveal some of the factors involved by allowing the identification of a broad scale of genes altered by seizures. In this study we used microarray analysis to reveal the gene expression profile of the laser microdissected hippocampal CA1 subregion one week after kainic acid (KA)-induced status epilepticus (SE) in 21-day-old rats, which are developmentally roughly comparable to juvenile children. The gene expression analysis with the Chipster software generated a total of 1592 differently expressed genes in the CA1 subregion of KA-treated rats compared to control rats. The KEGG database revealed that the identified genes were involved in pathways such as oxidative phosporylation (26 genes changed), and long-term potentiation (LTP; 18 genes changed). Also genes involved in Ca2+ homeostasis, gliosis, inflammation, and GABAergic transmission were altered. To validate the microarray results we further examined the protein expression for a subset of selected genes, glial fibrillary protein (GFAP), apolipoprotein E (apo E), cannabinoid type 1 receptor (CB1), Purkinje cell protein 4 (PEP-19), and interleukin 8 receptor (CXCR1), with immunohistochemistry, which confirmed the transcriptome results. Our results showed that SE resulted in no obvious CA1 neuronal loss, and alterations in the expression pattern of several genes during the early epileptogenic phase were comparable to previous gene expression studies of the adult hippocampus of both experimental epileptic animals and patients with temporal lobe epilepsy (TLE). However, some changes seem to occur after SE specifically in the juvenile rat hippocampus. Insight of the SE-induced alterations in gene expression and their related pathways could give us hints for the development of new target-specific antiepileptic drugs that interfere with the progression of the disease in the juvenile age group

Clamp loader ATPases and the evolution of DNA replication machinery

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life

Positive outcomes: validity, reliability and responsiveness of a novel person-centred outcome measure for people with HIV

Objectives Despite successful treatment, people living with HIV experience persisting and burdensome multidimensional problems. We aimed to assess the validity, reliability and responsiveness of Positive Outcomes, a patient-reported outcome measure for use in clinical practice. Methods In all, 1392 outpatients in five European countries self-completed Positive Outcomes, PAM-13 (patient empowerment), PROQOL-HIV (quality of life) and FRAIL (frailty) at baseline and 12 months. Analysis assessed: (a) validity (structural, convergent and divergent, discriminant); (b) reliability (internal consistency, test-retest); and (c) responsiveness. Results An interpretable four-factor structure was identified: ‘emotional wellbeing’, ‘interpersonal and sexual wellbeing’, ‘socioeconomic wellbeing’ and ‘physical wellbeing’. Moderate to strong convergent validity was found for three subscales of Positive Outcomes and PROQOL (ρ = −0.481 to −0.618, all p < 0.001). Divergent validity was found for total scores with weak ρ (−0.295, p < 0.001). Discriminant validity was confirmed with worse Positive Outcomes score associated with increasing odds of worse FRAIL group (4.81-fold, p < 0.001) and PAM-13 level (2.28-fold, p < 0.001). Internal consistency for total Positive Outcomes and its factors exceeded the conservative α threshold of 0.6. Test-retest reliability was established: those with stable PAM-13 and FRAIL scores also reported median Positive Outcomes change of 0. Improved PROQOL-HIV score baseline to 12 months was associated with improved Positive Outcomes score (r = −0.44, p < 0.001). Conclusions Positive Outcomes face and content validity was previously established, and the remaining validity, reliability and responsiveness properties are now demonstrated. The items within the brief 22-item tool are designed to be actionable by health and social care professionals to facilitate the goal of person-centred care