An official website of the United States government
Abstract Mass spectrometry (MS)‐based top‐down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post‐translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high‐resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS‐PAGE‐based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI‐MS) with capillary zone electrophoresis (CZE)‐MS/MS for high‐resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS‐PAGE gel and follow‐up cleanup as well as CZE‐MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high‐resolution separation and characterization of histone proteoforms. SDS‐PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high‐resolution separations of SDS‐PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.
more »
« less
Protein unfolding by SDS: the microscopic mechanisms and the properties of the SDS-protein assembly
The effects of detergent sodium dodecyl sulfate (SDS) on protein structure and dynamics are fundamental to the most common laboratory technique used to separate proteins and determine their molecular weights: polyacrylamide gel electrophoresis. However, the mechanism by which SDS induces protein unfolding and the microstructure of protein–SDS complexes remain largely unknown. Here, we report a detailed account of SDS-induced unfolding of two proteins—I27 domain of titin and β-amylase—obtained through all-atom molecular dynamics simulations. Both proteins were found to spontaneously unfold in the presence of SDS at boiling water temperature on the time scale of several microseconds. The protein unfolding was found to occur via two distinct mechanisms in which specific interactions of individual SDS molecules disrupt the protein's secondary structure. In the final state of the unfolding process, the proteins are found to wrap around SDS micelles in a fluid necklace-and-beads configuration, where the number and location of bound micelles changes dynamically. The global conformation of the protein was found to correlate with the number of SDS micelles bound to it, whereas the number of SDS molecules directly bound to the protein was found to define the relaxation time scale of the unfolded protein. Our microscopic characterization of SDS–protein interactions sets the stage for future refinement of SDS–enabled protein characterization methods, including protein fingerprinting and sequencing using a solid-state nanopore.
more »
« less
- PAR ID:
- 10170291
- Date Published:
- Journal Name:
- Nanoscale
- Volume:
- 12
- Issue:
- 9
- ISSN:
- 2040-3364
- Page Range / eLocation ID:
- 5422 to 5434
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Structural information of protein–protein interactions is essential for characterization of life processes at the molecular level. While a small fraction of known protein interactions has experimentally determined structures, computational modeling of protein complexes (protein docking) has to fill the gap. TheDockgroundresource (http://dockground.compbio.ku.edu) provides a collection of datasets for the development and testing of protein docking techniques. Currently,Dockgroundcontains datasets for the bound and the unbound (experimentally determined and simulated) protein structures, model–model complexes, docking decoys of experimentally determined and modeled proteins, and templates for comparative docking. TheDockgroundbound proteins dataset is a core set, from which otherDockgrounddatasets are generated. It is devised as a relational PostgreSQL database containing information on experimentally determined protein–protein complexes. This report on theDockgroundresource describes current status of the datasets, new automated update procedures and further development of the core datasets. We also present a newDockgroundinteractive web interface, which allows search by various parameters, such as release date, multimeric state, complex type, structure resolution, and so on, visualization of the search results with a number of customizable parameters, as well as downloadable datasets with predefined levels of sequence and structure redundancy.more » « less
-
Achieving reversible and tunable assembly of silica nanoparticles at liquid–liquid interfaces is vital for a wide range of scientific and technological applications including sustainable subsurface energy applications, catalysis, drug delivery and material synthesis. In this study, we report the mechanisms controlling the assembly of silica nanoparticles (dia. 50 nm and 100 nm) at water–heptane and water–toluene interfaces using sodium dodecyl sulfate (SDS) surfactant with concentrations ranging from 0.001–0.1 wt% using operando ultrasmall/small-angle X-ray scattering, cryogenic scanning electron microscopy imaging and classical molecular dynamics simulations. The results show that the assembly of silica nanoparticles at water–hydrocarbon interfaces can be tuned by controlling the concentrations of SDS. Silica nanoparticles are found to: (a) dominate the interfaces in the absence of interfacial SDS molecules, (b) coexist with SDS at the interfaces at low surfactant concentration of 0.001 wt% and (c) migrate toward the aqueous phase at a high SDS concentration of 0.1 wt%. Energetic analyses suggest that the van der Waals and electrostatic interactions between silica nanoparticles and SDS surfactants increase with SDS concentration. However, the favorable van der Waals and electrostatic interactions between the silica nanoparticles and toluene or heptane decrease with increasing SDS concentration. As a result, the silica nanoparticles migrate away from the water–hydrocarbon interface and towards bulk water at higher SDS concentrations. These calibrated investigations reveal the mechanistic basis for tuning silica nanoparticle assembly at complex interfaces.more » « less
-
Complex DNA topological structures, including polymer loops, are frequently observed in biological processes when protein molecules simultaneously bind to several distant sites on DNA. However, the molecular mechanisms of formation of these systems remain not well understood. Existing theoretical studies focus only on specific interactions between protein and DNA molecules at target sequences. However, the electrostatic origin of primary protein–DNA interactions suggests that interactions of proteins with all DNA segments should be considered. Here we theoretically investigate the role of non-specific interactions between protein and DNA molecules on the dynamics of loop formation. Our approach is based on analyzing a discrete-state stochastic model via a method of first-passage probabilities supplemented by Monte Carlo computer simulations. It is found that depending on a protein sliding length during the non-specific binding event three different dynamic regimes of the DNA loop formation might be observed. In addition, the loop formation time might be optimized by varying the protein sliding length, the size of the DNA molecule, and the position of the specific target sequences on DNA. Our results demonstrate the importance of non-specific protein–DNA interactions in the dynamics of DNA loop formations.more » « less
-
Binding-induced mechanical stabilization plays key roles in proteins involved in muscle contraction, cellular mechanotransduction, or bacterial adhesion. Because of the vector nature of force, single-molecule force spectroscopy techniques are ideal for measuring the mechanical unfolding of proteins. However, current approaches are still prone to calibration errors between experiments and geometrical variations between individual tethers. Here, we introduce a single-molecule assay based on magnetic tweezers and heterocovalent attachment, which can measure the binding of the substrate–ligand using the same protein molecule. We demonstrate this approach with protein L, a model bacterial protein which has two binding interfaces for the same region of kappa-light chain antibody ligands. Engineered molecules with eight identical domains of protein L between a HaloTag and a SpyTag were exposed to repeated unfolding–refolding cycles at forces up to 100 pN for several hours at a time. The unfolding behavior of the same protein was measured in solution buffers with different concentrations of antibody ligands. With increasing antibody concentration, an increasing number of protein L domains became more stable, indicative of ligand binding and mechanical reinforcement. Interestingly, the dissociation constant of the mechanically reinforced states coincides with that measured for the low-avidity binding interface of protein L, suggesting a physiological role for the second binding interface. The molecular approach presented here opens the road to a new type of binding experiments, where the same molecule can be exposed to different solvents or ligands.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c9nr09135a
