Protein Unfolding of metMyoglobin Monitored by Spectroscopic Techniques

In this experiment the unfolding of the protein, myoglobin, will be monitored using both fluorescence and UV-vis absorption spectroscopy. Changes in the absorbance at 409.5 nm, the absorption maximum of the native state, will be monitored in order to probe changes in the protein conformation after initiation of unfolding by addition of a chemical denaturant. Protein unfolding will also be monitored after exciting the sample at 280 nm and following protein fluorescence emission at 345 nm, the fluorescence maximum for the unfolded state. The absorption run follows the time-dependent decrease in the concentration of the native-state species, whereas the fluorescence experiment monitors the increase in the concentration of the unfolded state. Kinetic rate constants obtained using the two techniques will be compared.     

In Silico Evidence That Protein Unfolding is a Precursor of Protein Aggregation

We present a computational study on the folding and aggregation of proteins in an aqueous environment, as a function of its concentration. We show how the increase of the concentration of individual protein species can induce a partial unfolding of the native conformation without the occurrence of aggregates. A further increment of the protein concentration results in the complete loss of the folded structures and induces the formation of protein aggregates. We discuss the effect of the protein interface on the water fluctuations in the protein hydration shell and their relevance in the protein-protein interaction.     

Isoentropic and Isoenthalpic Temperatures of Protein Unfolding in Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is widely used for the separation and purification of biopolymers in their native state, and now also becomes a new method to refold the denatured proteins1. The effect of salt or water concentration2 on the pr…     

蛋白质非共价化合物的电喷雾质谱研究

介绍了近几年来利用电喷雾离子化质谱技术研究蛋白质的基本构象及蛋白质非共价化合物的基本化学信息的进展。许多研究表明,电喷雾质谱在研究蛋白质的复杂结构和功能方面有十分广阔的前景。文中列举了若干实例说明了电喷雾离子化质谱技术在分析蛋白质非共价化合物的应用和一般方法。     

电喷雾质谱的非共价键蛋白质复合物研究

电喷雾质谱(ESI-MS)已经成为检测和研究生物分子弱相互作用,即非共价键作用的一个重要分析手段.ESI-MS除了具有快速、灵敏、专属的特点以外,还有能够直接得出复合物的分子量和化学计量比的优点.本文通过蛋白质与蛋白质、配体、金属离子的非共价复合物的例子阐述了ESI-MS技术的主要特性,综述了ESI-MS在非共价键蛋白质复合物方面的早期和近期应用研究成果.引用文献34篇.     

β-环糊精与蛋白质非共价复合物的电喷雾质谱研究

电喷雾质谱(ESI-MS)已经广泛应用于非共价复合物的检测和研究。ESI是一种极软的离子化过程,它不仅能在不断裂共价键的情况下使分子离子化,而且可以在离子化过程中保持分子间弱的非共价键作用。因此ESI-MS在研究非共价键复合物方面有着独特的作用,并且ESI-MS还能提供复合物重要的化学计量学信息。环糊精(简写为CD)是一类被大家所熟知的化合物,它可以和许多客体分子在水溶液里形成包合复合物,更为重要的是环糊精是目前人工合成模拟酶研究中最好的模型之一。通过对环糊精与生物分子间形成的非共价键复合物的研究常常可以揭示出天然生物体     

An Unusual Protein–Protein Interaction through Coupled Unfolding and Binding

Aptides, a novel class of high‐affinity peptides, recognize diverse molecular targets with high affinity and specificity. The solution structure of the aptide APT specifically bound to fibronectin extradomain B (EDB), which represents an unusual protein–protein interaction that involves coupled unfolding and binding, is reported. APT binding is accompanied by unfolding of the C‐terminal β strand of EDB, thereby permitting APT to interact with the freshly exposed hydrophobic interior surfaces of EDB. The β‐hairpin scaffold of APT drives the interaction by a β‐strand displacement mechanism, such that an intramolecular β sheet is replaced by an intermolecular β sheet. The unfolding of EDB perturbs the tight domain association between EDB and FN8 of fibronectin, thus highlighting its potential use as a scaffold that switches between stretched and bent conformations.     

Comparing the Effects of Additives on Protein Analysis Between Desorption Electrospray (DESI) and Electrospray Ionization (ESI)

It is frequently said that DESI-MS follows a similar ionization mechanism as ESI because of similarities usually observed in their respective mass spectra. However, practical use of DESI-MS for protein analysis is limited to proteins with lower molecular weights (<?25 kDa) due to a mass-dependent loss in signal intensity. Here we investigated commonly used volatile acids and their ammonium salt buffers for DESI-MS analysis of protein. We noticed that, surprisingly, some additives influence the analysis differently in DESI compared to ESI. Improved signal intensities with both DESI and ESI were obtained when acetic and formic acid were added into aqueous methanol spray solvents with both DESI and ESI. On the other hand, while with ESI the addition of ammonium salts into spray solutions strongly reduced both signal and S/N, with DESI signal intensities and S/N were improved dramatically. Ammonium bicarbonate when used with DESI reduced the total amount of adduction and delivered excellent signal-to-noise ratios with high intensity; however, it also denatures protein. When native state protein mass spectra are preferred, ammonium acetate would also deliver reasonable adduct removal and improved S/N. The amount of total adduction of individual adducting species and of all species could not be correlated with differences in either solutions pH values or with proton affinities of the anions. An obvious difference between DESI and ESI mass spectrometry is the effects of protein solubility during droplet pickup (desorption), but differences in the sizes, velocities, and composition of ionizing droplets were also discussed as important factors.

Open image in new window

Graphical Abstract ?

     

A Computational Model for Protein Ionization by Electrospray Based on Gas-Phase Basicity

Identifying the key factor(s) governing the overall protein charge is crucial for the interpretation of electrospray-ionization mass spectrometry data. Current hypotheses invoke different principles for folded and unfolded proteins. Here, first we investigate the gas-phase structure and energetics of several proteins of variable size and different folds. The conformer and protomer space of these proteins ions is explored exhaustively by hybrid Monte-Carlo/molecular dynamics calculations, allowing for zwitterionic states. From these calculations, the apparent gas-phase basicity of desolvated protein ions turns out to be the unifying trait dictating protein ionization by electrospray. Next, we develop a simple, general, adjustable-parameter-free model for the potential energy function of proteins. The model is capable to predict with remarkable accuracy the experimental charge of folded proteins and its well-known correlation with the square root of protein mass.     

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.     

Insights into the Mechanism of Protein Electrospray Ionization From Salt Adduction Measurements

The mechanisms whereby protein ions are liberated from charged droplets during electrospray ionization (ESI) remain under investigation. Compact conformers electrosprayed from aqueous solution in positive ion mode likely follow the charged residue model (CRM), which envisions analyte release after solvent evaporation to dryness. The concentration of nonvolatile salts such as NaCl increases sharply within vanishing CRM droplets, promoting nonspecific pairing of Cl^- and Na⁺ with charged groups on the protein surface. For unfolded proteins, it has been proposed that ion formation occurs via the chain ejection model (CEM). During the CEM proteins are expelled from the droplet long before complete solvent evaporation has taken place. Here we examine whether salt adduction levels support the view that folded and unfolded proteins follow different ESI mechanisms. Solvent evaporation during the CEM is expected to be less extensive and, hence, the salt concentration at the point of protein release should be substantially lower than for the CRM. CEM ions should therefore exhibit lower adduction levels than CRM species. We explore the adduction behavior of several proteins that were chosen to allow comparative studies on folded and unfolded structures in the same solution. In-source activation eliminates chloride adducts via HCl release, generating protein ions that are heterogeneously charged because of sodiation and protonation. Sodiation levels measured under such conditions provide estimates of the salt adduction behavior experienced by the “nascent” analyte ions. Sodiation levels are significantly reduced for unfolded proteins, supporting the view that these species are indeed formed via the CEM.

Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding

Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap‐closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β‐barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped‐flow experiments indicate that a rate‐limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H‐D exchange data demonstrate the presence of two excited states: one is native‐like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate.     

In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry

Here, an electrokinetic extraction (EkE) syringe is presented allowing for on-line electrokinetic removal of serum proteins before ESI-MS. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5× dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in-syringe. Signal enhancements of 3.6–32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE-ESI-MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng mL⁻¹ for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs, the EkE-ESI-MS LOQs were 3.1 μg mL⁻¹ and 2.9 μg mL⁻¹ for naproxen and paracetamol, respectively. The EkE-ESI-MS and EkE-ESI-MS/MS methods showed good accuracy (%found of 81 % to 120 %), precision (≤20 %), and linearity (r>0.997) for all the studied drugs in spiked serum samples.     

Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

Electron capture dissociation was used to probe the structure, unfolding, and folding of KIX ions in the gas phase. At energies for vibrational activation that were sufficiently high to cause loss of small molecules such as NH₃ and H₂O by breaking of covalent bonds in about 5% of the KIX (M + nH)ⁿ⁺ ions with n = 7–9, only partial unfolding was observed, consistent with our previous hypothesis that salt bridges play an important role in stabilizing the native solution fold after transfer into the gas phase. Folding of the partially unfolded ions on a timescale of up to 10 s was observed only for (M + nH)ⁿ⁺ ions with n = 9, but not n = 7 and n = 8, which we attribute to differences in the distribution of charges within the (M + nH)ⁿ⁺ ions.

Open image in new window

Graphical Abstract ?

     

In Situ Monitored Vortex Fluidic-Mediated Protein Refolding/Unfolding Using an Aggregation-Induced Emission Bioprobe

Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for β-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with β-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.     

What Protein Charging (and Supercharging) Reveal about the Mechanism of Electrospray Ionization

Understanding the charging mechanism of electrospray ionization is central to overcoming shortcomings such as ion suppression or limited dynamic range, and explaining phenomena such as supercharging. Towards that end, we explore what accumulated observations reveal about the mechanism of electrospray. We introduce the idea of an intermediate region for electrospray ionization (and other ionization methods) to account for the facts that solution charge state distributions (CSDs) do not correlate with those observed by ESI-MS (the latter bear more charge) and that gas phase reactions can reduce, but not increase, the extent of charging. This region incorporates properties (e.g., basicities) intermediate between solution and gas phase. Assuming that droplet species polarize within the high electric field leads to equations describing ion emission resembling those from the equilibrium partitioning model. The equations predict many trends successfully, including CSD shifts to higher m/z for concentrated analytes and shifts to lower m/z for sprays employing smaller emitter opening diameters. From this view, a single mechanism can be formulated to explain how reagents that promote analyte charging (“supercharging”) such as m-NBA, sulfolane, and 3-nitrobenzonitrile increase analyte charge from “denaturing” and “native” solvent systems. It is suggested that additives’ Brønsted basicities are inversely correlated to their ability to shift CSDs to lower m/z in positive ESI, as are Brønsted acidities for negative ESI. Because supercharging agents reduce an analyte’s solution ionization, excess spray charge is bestowed on evaporating ions carrying fewer opposing charges. Brønsted basicity (or acidity) determines how much ESI charge is lost to the agent (unavailable to evaporating analyte). Graphical Abstract

?     

Effects of Cations on Protein and Peptide Charging in Electrospray Ionization from Aqueous Solutions

The effects of eight different cations with ionic radii between 69 and 337 pm on the charging of peptides and proteins with electrospray ionization from aqueous acetate salt solutions are reported. Significant adduction occurs for all cations except NH₄ ⁺, and the average protein charge is lower when formed from solutions containing salts compared with solutions without salts added. Circular dichroism and ion mobility results show the protein conformations are different in pure water compared with salt solutions, which likely affects the extent of charging. The average charge of protein and peptide ions formed from solutions with Li⁺ and Cs⁺, which have Gibbs solvation free energies (GSFEs) that differ by 225 kJ/mol, is similar. Lower charge states are typically formed from solutions with tetramethylammonium and tetraethylammonium that have lower GSFE values. Loss of the larger cations that have the lowest GSFEs is facile when adducted protein ions are collisionally activated, resulting in the formation of lower analyte charge states. This reaction pathway provides a route to produce abundant singly protonated protein ions under native mass spectrometry conditions. The average protein and peptide charge with NH₄ ⁺ is nearly the same as that with Rb⁺ and K⁺, cations with similar GSFE and ionic radii. This indicates that proton transfer from NH₄ ⁺ to proteins plays an insignificant role in the extent of protein charging in native mass spectrometry.

Elucidating Factors Manipulated the Formation of Multiply Charged Protein Homomultimeric Complexes by Electrospray Ionization

Native non‐covalently bonded protein‐protein and protein‐substrate complexes are of great interest and have been extensively studied by electrospray ionization mass spectrometry (ESI‐MS). Multiply charged protein homomultimeric complexes are shown to form by ESI‐MS. This study addresses factors that can artificially induce the formation of multiply charged protein homomultimeric complexes. Cytochrome c (Cyt c) and ubiquitin, which are monomers in solution, were found to generate (Cyt c)_mⁿ⁺ by electrospray ionization (ESI). The homomultimeric complexes were not limited to dimeric complexes but include also multiply charged trimers, tetramers, and pentamers. The observation of these homomultimeric complexes has never been revealed from a Cyt c solution at the concentration as low as 10 μM. Increasing the concentration of Cyt c enhanced the formation of (Cyt c)_mⁿ⁺ as expected; however, the protein concentration does not affect the relative intensities of monomeric and dimeric complexes. Additionally the enrichment of NH₄OH also promotes the formation of (Cyt c)_mⁿ⁺. Notably, source collision‐induced dissociations (source‐CID) of (Cyt c)_mⁿ⁺ alter the charge state distribution (CSD) and may lead to an incorrect interpretation of Cyt c conformations. Hence, extra care should be taken when using CSD to interpret the conformation of a protein derived from ESI‐MS.