Linkage between fitness of yeast cells and adenylate kinase catalysis

Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies

Guidelines for the assessment of bone density and microarchitecture in vivo using high-resolution peripheral quantitative computed tomography

IntroductionThe application of high-resolution peripheral quantitative computed tomography (HR-pQCT) to assess bone microarchitecture has grown rapidly since its introduction in 2005. As the use of HR-pQCT for clinical research continues to grow, there is an urgent need to form a consensus on imaging and analysis methodologies so that studies can be appropriately compared. In addition, with the recent introduction of the second-generation HrpQCT, which differs from the first-generation HR-pQCT in scan region, resolution, and morphological measurement techniques, there is a need for guidelines on appropriate reporting of results and considerations as the field adopts newer systems.MethodsA joint working group between the International Osteoporosis Foundation, American Society of Bone and Mineral Research, and European Calcified Tissue Society convened in person and by teleconference over several years to produce the guidelines and recommendations presented in this document.ResultsAn overview and discussion is provided for (1) standardized protocol for imaging distal radius and tibia sites using HR-pQCT, with the importance of quality control and operator training discussed; (2) standardized terminology and recommendations on reporting results; (3) factors influencing accuracy and precision error, with considerations for longitudinal and multi-center study designs; and finally (4) comparison between scanner generations and other high-resolution CT systems.ConclusionThis article addresses the need for standardization of HR-pQCT imaging techniques and terminology, provides guidance on interpretation and reporting of results, and discusses unresolved issues in the field

Coexistence of multiple minor states of fatty acid binding protein and their functional relevance

Proteins are dynamic over a wide range of timescales, but determining the number of distinct dynamic processes and identifying functionally relevant dynamics are still challenging. Here we present the study on human intestinal fatty acid binding protein (hIFABP) using a novel analysis of (15)N relaxation dispersion (RD) and chemical shift saturation transfer (CEST) experiments. Through combined analysis of the two types of experiments, we found that hIFABP exists in a four-state equilibrium in which three minor states interconvert directly with the major state. According to conversion rates from the major “closed” state to minor states, these minor states are irrelevant to the function of fatty acid transport. Based on chemical shifts of the minor states which could not be determined from RD data alone but were extracted from a combined analysis of RD and CEST data, we found that all the minor states are native-like. This conclusion is further supported by hydrogen-deuterium exchange experiments. Direct conversions between the native state and native-like intermediate states may suggest parallel multitrack unfolding/folding pathways of hIFABP. Moreover, hydrogen-deuterium exchange data indicate the existence of another locally unfolded minor state that is relevant to the fatty acid entry process

Analysing persistent organic pollutants in eggs, blood and tissue of the green sea turtle (Chelonia mydas) using gas chromatography with tandem mass spectrometry (GC-MS/MS)

Investigation into persistent organic pollutants (POPs) in sea turtles is an important area of conservation research due to the harmful effects of these chemicals. However, the analysis of POPs in the green sea turtle (Chelonia mydas) has been limited by methods with relatively high limits of detection and high costs associated with multiple sample injections into complex arrangements of analytical equipment. The present study aimed to develop a method that could detect a large number of POPs in the blood, eggs and tissue of C. mydas at trace concentrations. A gas chromatography with tandem mass spectrometry (GC-MS/MS) method was developed that could report 125 POP compounds to a limit of detection o