Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell , 2003 , 12 , 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Δ G ) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilised by up to 6 kcal mol −1 relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Δ G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Δ G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56088/1/1162_ftp.pd

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

©2009 Gao, Skolnick. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.doi:10.1371/journal.pcbi.1000341DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Ca deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein

Technical note: Lessons from and best practices for the deployment of the Soil Water Isotope Storage System

Soil water isotope datasets are useful for understanding connections between the hydrosphere, atmosphere, biosphere, and geosphere. However, they have been underproduced because of the technical challenges associated with collecting those datasets. Here, we present the results of testing and automation of the Soil Water Isotope Storage System (SWISS). The unique innovation of the SWISS is that we are able to automatically collect water vapor from the critical zone at a regular time interval and then store that water vapor until it can be measured back in a laboratory setting. Through a series of quality assurance and quality control tests, we tested whether the SWISS is resistant to both atmospheric intrusion and leaking in both laboratory and field settings. We assessed the accuracy and precision of the SWISS through a series of experiments in which water vapor of known composition was introduced into the flasks, stored for 14 d, and then measured. From these experiments, after applying an offset correction to report our values relative to Vienna Standard Mean Ocean Water (VSMOW), we assess the precision of the SWISS to be ±0.9 ‰ and ±3.7 ‰ for δ18O and δ2H, respectively. We deployed three SWISS units at three different field sites to demonstrate that the SWISS stores water vapor reliably enough that we are able to differentiate dynamics both between the sites as well within a single soil column. Overall, we demonstrate that the SWISS retains the stable isotope composition of soil water vapor for long enough to allow researchers to address a wide range of ecohydrologic questions.</p

The Time Structure of Hadronic Showers in highly granular Calorimeters with Tungsten and Steel Absorbers

The intrinsic time structure of hadronic showers influences the timing capability and the required integration time of hadronic calorimeters in particle physics experiments, and depends on the active medium and on the absorber of the calorimeter. With the CALICE T3B experiment, a setup of 15 small plastic scintillator tiles read out with Silicon Photomultipliers, the time structure of showers is measured on a statistical basis with high spatial and temporal resolution in sampling calorimeters with tungsten and steel absorbers. The results are compared to GEANT4 (version 9.4 patch 03) simulations with different hadronic physics models. These comparisons demonstrate the importance of using high precision treatment of low-energy neutrons for tungsten absorbers, while an overall good agreement between data and simulations for all considered models is observed for steel.Comment: 24 pages including author list, 9 figures, published in JINS

Pion and proton showers in the CALICE scintillator-steel analogue hadron calorimeter

Showers produced by positive hadrons in the highly granular CALICE scintillator-steel analogue hadron calorimeter were studied. The experimental data were collected at CERN and FNAL for single particles with initial momenta from 10 to 80 GeV/c. The calorimeter response and resolution and spatial characteristics of shower development for proton- and pion-induced showers for test beam data and simulations using Geant4 version 9.6 are compared.Comment: 26 pages, 16 figures, JINST style, changes in the author list, typos corrected, new section added, figures regrouped. Accepted for publication in JINS

Shower development of particles with momenta from 15 GeV to 150 GeV in the CALICE scintillator-tungsten hadronic calorimeter

We present a study of showers initiated by electrons, pions, kaons, and protons with momenta from 15 GeV to 150 GeV in the highly granular CALICE scintillator-tungsten analogue hadronic calorimeter. The data were recorded at the CERN Super Proton Synchrotron in 2011. The analysis includes measurements of the calorimeter response to each particle type as well as measurements of the energy resolution and studies of the longitudinal and radial shower development for selected particles. The results are compared to Geant4 simulations (version 9.6.p02). In the study of the energy resolution we include previously published data with beam momenta from 1 GeV to 10 GeV recorded at the CERN Proton Synchrotron in 2010.Comment: 35 pages, 21 figures, 8 table

Performance of the first prototype of the CALICE scintillator strip electromagnetic calorimeter

A first prototype of a scintillator strip-based electromagnetic calorimeter was built, consisting of 26 layers of tungsten absorber plates interleaved with planes of 45x10x3 mm3 plastic scintillator strips. Data were collected using a positron test beam at DESY with momenta between 1 and 6 GeV/c. The prototype's performance is presented in terms of the linearity and resolution of the energy measurement. These results represent an important milestone in the development of highly granular calorimeters using scintillator strip technology. This technology is being developed for a future linear collider experiment, aiming at the precise measurement of jet energies using particle flow techniques