Degradation Parameters from Pulse-Chase Experiments

2000

Biomedical Optics Express, 2011

Fluorescence lifetime imaging has emerged as an important microscopy technique, where high repetition rate lasers are the primary light sources. As fluorescence lifetime becomes comparable to intervals between consecutive excitation pulses, incomplete fluorescence decay from previous pulses can superimpose onto the subsequent decay measurements. Using a mathematical model, the incomplete decay effect has been shown to lead to overestimation of the amplitude average lifetime except in monoexponential decays. An inverse model is then developed to correct the error from this effect and the theoretical simulations are tested by experimental results.

Biophysical Journal, 1995

Considerable effort in instrument development has made possible detection of picosecond fluorescence lifetimes by time-correlated single-photon counting. In particular, efforts have been made to narrow markedly the instrument response function (IRF). Less attention has been paid to analytical methods, especially to problem of discretization of the convolution integral, on which the detection and quantification of short lifetimes critically depends. We show that better discretization methods can yield acceptable results for short lifetimes even with an IRF several times wider than necessary for the standard discretization based on linear approximation (LA). A general approach to discretization, also suitable for nonexponential models, is developed. The zero-time shift is explicitly included. Using simulations, we compared LA, quadratic, and cubic approximations. The latter two proved much better for detection of short lifetimes and, in that respect, they do not differ except when the zero-time shift exceeds two channels, when one can benefit from using the cubic approximation. We showed that for LA in some cases narrowing the IRF beyond FWHM = 150 ps is actually counterproductive. This is not so for quadratic and cubic approximations, which we recommend for general use.

Chemistry & Biology, 2011

Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Here, we demonstrate how chemical labeling of SNAP-tag fusion proteins can be exploited to measure the half-life of resident intracellular and extracellular proteins in living mice. First, we demonstrate that SNAP-tag substrates have wide bioavailability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then apply near-infrared probes to perform noninvasive imaging of in vivo-labeled tumors. Finally, we use SNAP-mediated chemical pulse-chase labeling to perform measurement of the in vivo half-life of different extra-and intracellular proteins. These results open broad perspectives for studying protein function in living animals.

Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance and reaching ‘undruggable’ proteins compared to traditional small molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation, instead one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs) understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The Cellular Thermal Shift Assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring comp...

Archives of Biochemistry and Biophysics, 1975

Several case studies have suggested that low-latitude Pi2 pulsations are triggered or driven by earthward flow bursts in the magnetotail. Until now, no statistical study has investigated the causality between the flow bursts and Pi2 pulsations, so the overall significance of flow bursts to Pi2 excitation remains unclear. In this study we statistically examine the relationship between earthward flow bursts and low-latitude Pi2 pulsations. We address the following two questions: (1) Does every flow burst trigger a low-latitude Pi2 pulsation? (2) Does every low-latitude Pi2 pulsation have a corresponding flow burst? We use magnetic field and ion plasma measurements made by Geotail and Pi2 measurements made at Kakioka from January 1995 to July 2001. Regarding the first question, we identified 849 Geotail flow burst events in the near-Earth magnetotail and found that $90% of them occurred in the region characterized by jB x j < 10 nT, and $75% occurred in the region characterized by jB x j < 5 nT (i.e., at locations very close to the midplane of the plasma sheet). We found that 31 Pi2 events occurred within the 10-min interval after the peak velocity of a flow burst; that is, only $4% of the flow burst events produced a low-latitude Pi2. Regarding the second question, we identified 556 Pi2 events at Kakioka at times when Geotail was in the near-Earth magnetotail, and of these, 31 were the subset of the 849 flow bursts identified above. This gives a seemingly low ($6%) association of flow burst with Pi2. However, we find that the association rate jumped to $26% when Geotail was in the region of jB x j < 5 nT, implying that there is a great chance of detecting a flow burst at the time of a ground Pi2 if plasma measurements are made very close to the midplane of the plasma sheet. We conclude that only a small fraction of plasma sheet flow bursts generate Pi2 pulsations, while each Pi2 likely has an associated flow burst somewhere in the near-Earth magnetotail.

2020

Isotopic labeling with deuterium oxide (D2O) is a common technique for estimatingin vivoprotein turnover, but its use has been limited by two long-standing problems: (1) identifying non-monoisotopic peptides; and (2) estimating protein turnover rates in the presence of dynamic amino acid enrichment. In this paper, we present a novel experimental and analytical framework for solving these two problems. Peptides with high probabilities of labeling in many amino acids present fragmentation spectra that frequently do not match the theoretical spectra used in standard identification algorithms. We resolve this difficulty using a modified search algorithm we call Conditional Ion Distribution Search (CIDS). Increased identifications from CIDS along with direct measurement of amino acid enrichment and statistical modeling that accounts for heterogeneous information across peptides, dramatically improves the accuracy and precision of half-life estimates. We benchmark the approach in cells, w...

Journal of Photochemistry and Photobiology B: Biology, 2013

Tryptophan is the most often investigated intrinsic fluorophore due to its abundance in proteins and its sensitivity to different environmental conditions. Fluorescence quenching is a powerful method to study proteins and acrylamide is a frequently applied quencher in these investigations. Quenching experiments are sometimes distorted by the undesired protein-quencher interactions that can result in a misinterpretation of the results. Here we focused on the identification of the possible side-effects of acrylamide applying fluorescence lifetime measurements. To provide reference data for protein denaturation the fluorescence parameters were also recorded in the presence of different concentrations of guanidine hydrochloride. In circular dichroism experiments we characterized directly the acrylamide effect on the tertiary structure of the proteins. According to the obtained data in experiments with seven tryptophan-containing proteins the full width at half maximum (FWHM) of the fluorescence lifetime distribution is an appropriate parameter to monitor the undesired effects of acrylamide on the proteins.

Bioinformatics, 2014

Motivation: Fluorescence recovery after photobleaching (FRAP) is a functional live cell imaging technique that permits the exploration of protein dynamics in living cells. To extract kinetic parameters from FRAP data, a number of analytical models have been developed. Simplifications are inherent in these models, which may lead to inexhaustive or inaccurate exploitation of the experimental data. An appealing alternative is offered by the simulation of biological processes in realistic environments at a particle level. However, inference of kinetic parameters using simulation-based models is still limited. Results: We introduce and demonstrate a new method for the inference of kinetic parameter values from FRAP data. A small number of in silico FRAP experiments is used to construct a mapping from FRAP recovery curves to the parameters of the underlying protein kinetics. Parameter estimates from experimental data can then be computed by applying the mapping to the observed recovery curves. A bootstrap process is used to investigate identifiability of the physical parameters and determine confidence regions for their estimates. Our method circumvents the computational burden of seeking the best-fitting parameters via iterative simulation. After validation on synthetic data, the method is applied to the analysis of the nuclear proteins Cdt1, PCNA and GFPnls. Parameter estimation results from several experimental samples are in accordance with previous findings, but also allow us to discuss identifiability issues as well as cell-to-cell variability of the protein kinetics. Implementation: All methods were implemented in MATLAB R2011b. Monte Carlo simulations were run on the HPC cluster Brutus of ETH

Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment

erably less than the current best estimate of (880.1 ± 1.1) s promulgated by the Particle Data Group [1]. Currently, experimental studies are underway to determine if this discrepancy can be explained by neutron capture by 3 He impurities in the trapping volume. We also quantify uncertainties associated with Monte Carlo sampling variability and imperfect knowledge of physical models for neutron interactions with materials at the walls of trap as well as beam divergence effects. Finally, in a Monte Carlo experiment, we demonstrate that when the trapping potential is ramped down and then back up again, systematic error due to wall losses of marginally trapped neutrons can be suppressed to a very low level. Monte Carlo simulation studies indicate that this ramping strategy is more efficient than an alternative ramping scheme where UCNs are produced when the field is fully ramped, and then increased to its maximum value after the trap is filled. The survival probability formalism developed here should be applicable to other experiments where neutron (or other particle) loss mechanisms are non-trivial (i.e., where the associated survival probability function with the loss mechanism is non-exponential).