Native denaturation differential scanning fluorimetry: Determining the effect of urea using a quantitative real-time thermocycler

The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (K_nd), and protein unfolding by urea (I₅₀). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity.     

荧光热漂移实验体外检测核小体的解聚

核小体是真核生物染色质的基本组成单元。核小体的解聚可以动态调控诸如DNA复制、转录、重组等以DNA为模板的生物学过程。特定荧光探针分子与蛋白质疏水残基结合后可被激发荧光信号。荧光热漂移（fluorescence thermal shift,FTS）是一种利用该原理检测温度上升时蛋白质变性过程的方法。本文以Widom 601序列为DNA模板,盐透析法体外装配核小体;分别在实时荧光定量PCR仪的VIC和TAMRA通道激发下,利用FTS检测了核小体在Tris-NaCl（TN）缓冲液和HEPES缓冲液中的解聚状态。研究结果发现,DNA对照序列在TN缓冲液中产生的背景噪声较大,VIC通道激发下组蛋白八聚体产生的荧光信号相对较强。FTS检测核小体的结果显示,随着温度的上升核小体的解聚过程分为2个阶段,～71℃和～84℃是核小体解聚速度最快的温度范围,而75℃～80℃是降解速度较慢的阶段。基于FTS基本原理,本研究发展了一种新颖的可用于检测核小体结构稳定性的方法,为核小体与染色质结构相关问题的研究奠定了一定的技术基础。     

Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies by up‐converting phosphor technology‐based lateral‐flow assay

In this study, an up‐converting phosphor technology‐based lateral‐flow (UPT‐LF) assay was developed to detect severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies rapidly and specifically. SFTSV recombinant N protein (SFTSV‐rNP) was coated on analytical membrane for sample capture, up‐converting phosphor (UCP) particles were used as the reporter, the luminescence emitted by UCP particles was converted to a measurable signal by a biosensor. The performance of UPT‐LF assay was evaluated by testing 302 field serum samples by ELISA (enzyme‐linked immunosorbent assay), Western blotting and UPT‐LF assay. UPT‐LF assay exhibited a lower detection limit than ELISA, and a satisfied level of agreement was exhibited by Kappa statistics (Kappa coefficient = 0.938). Considering Western blotting as the reference for comparison, the sensitivity and specificity of UPT‐LF assay could reach 98.31% and 100%. UPT‐LF assay showed no specific reaction with hantavirus total serum antibodies, which avoids the misdiagnosis of SFTSV from hantavirus that could cause similar clinical symptoms. UPT‐LF assay was able to achieve acceptable results within 15 min and needed only 10 μL sample for each test. As a whole, UPT‐LF assay is a candidate method for on‐site surveillance of SFTSV total antibodies owing to its excellent sensitivity, specificity, stability, easy operation and for being less time consuming.     

Investigating protein unfolding kinetics by pulse proteolysis

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (K_d) of the complex from unfolding kinetic constants, which is also in good agreement with known K_d values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.     

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa‐histidine tag, all attached to the C‐terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C‐ or N‐terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His₆ tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Revisiting absorbance at 230 nm as a protein unfolding probe

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A₂₃₀) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A₂₃₀ as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A₂₃₀. Significant changes in A₂₃₀ were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A₂₃₀ in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A₂₃₀ under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A₂₃₀ allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A₂₃₀ is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.     

Direct colorimetric assay of microcystin using protein phosphatase

A new direct colorimetric assay of microcystin in water and algal samples is proposed consisting of two procedures as follows: 1) the elimination of phosphorus in the sample and concentration of microcystin using a C₁₈ cartridge, 2) the detection of the released phosphorus by the ascorbic acid method and determination of protein phosphatase (PP) inhibition by microcystin. The optimum amounts of phosphorylase α and PP-1 in 50 μL concentrated sample were 50 μg/50 μL buffer and 1.0 unit/50 μL buffer, respectively, for the best assay. The pH for the maximum activity of PP-1 was 8. The minimum detectable concentration for this method was about 0.02 μg/L, which is sufficient to meet the proposed guideline level of 1 μg microcystin/L in drinking water. Consequently, it would seem that the proposed direct colorimetric assay using PP is a rapid, easy, and convenient method for the detection of microcystin in water and algal samples.     

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their β‐turn occurrence, we engineered two chimerical enzymes where their super secondary β‐loop‐α motifs 2 ((βα)₂) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (βα)₂ motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα)₂ motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (βα)₂ of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N‐terminal of loop‐2 and the C‐terminal of α‐helix‐2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (E_A). A systematic analysis of DSC data showed a large decrease on the E_A of TcTIM (ΔE_A ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571–579. © 2016 Wiley Periodicals, Inc.     

Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward beta-sheet structure

The term kinetic stability is used to describe proteins that are trapped in a specific conformation because of an unusually high-unfolding barrier that results in very slow unfolding rates. Motivated by the observation that some proteins are resistant to sodium dodecyl sulfate (SDS)-induced denaturation, an attempt was made to determine whether this property is a result of kinetic stability. We studied many proteins, including a few kinetically stable proteins known to be resistant to SDS. The resistance to SDS-induced denaturation was investigated by comparing the migration on polyacrylamide gels of identical boiled and unboiled protein samples containing SDS. On the basis of the different migration of these samples, eight proteins emerged as being resistant to SDS. The kinetic stability of these proteins was confirmed by their slow unfolding rate upon incubation in guanidine hydrochloride. Further studies showed that these proteins were also extremely resistant to proteolysis by proteinase K, suggesting that a common mechanism may account for their resistance to SDS and proteolytic cleavage. Together, these observations suggest that a rigid protein structure may be the physical basis for kinetic stability and that resistance to SDS may serve as a simple assay for identifying proteins whose native conformations are kinetically trapped. Remarkably, most of the kinetically stable SDS-resistant proteins in this study are oligomeric beta-sheet proteins, suggesting a bias of these types of structures toward kinetic stability.     

A one-pot analysis approach to simplify measurements of protein stability and folding kinetics

To achieve a good understanding of the characteristics of a protein, it is important to study its stability and folding kinetics. Investigations of protein stability have been recently applied to drug-target identification, drug screening, and proteomic studies. The efficiency of the experiments performed to study protein stability and folding kinetics is now a crucial factor that needs to be optimized for these potential applications. However, the standard procedures used to carry out these experiments are usually complicated and time consuming. Large number of measurements is the bottleneck that limits the application of protein folding to large-scale experiments. To overcome this limitation, we developed a method denoted as “one-pot analysis” which is based on taking a single measurement from a mixture of samples rather than from every sample. We combined one-pot analysis with pulse proteolysis to determine the effects of the binding of maltose to maltose-binding protein on the protein folding properties. After carrying out a simple optimization, we demonstrated that protein stability or unfolding kinetics could be measured accurately with just one detection measurement. We then further applied the optimized conditions to cellular thermal shift assay (CETSA). Combining one-pot analysis with CETSA led to a successful determination of the effects of the binding of methotrexate to dihydrofolate reductase in HCT116 cancer cells. Our results demonstrated the applicability of one-pot analysis to energetics-based methods for studying protein folding. We expect the combination of one-pot analysis and energetics-based methods to significantly benefit studies such as drug-target identification, proteomic investigations, and drug screening.     

Surfactant-induced unfolding of cellulase: kinetic studies

Surfactant-induced unfolding is a significant degradation pathway for detergent enzymes. This study examines the kinetics of surfactant-induced unfolding for endoglucanase III, a detergent cellulase, under conditions of varying pH, temperature, ionic strength, surfactant type, and surfactant concentration. Interactions between protein and surfactant monomer are shown to play a key role in determining the kinetics of the unfolding process. We demonstrate that the unfolding rate can be slowed by (1) modifying protein charge and/or pH conditions to create electrostatic repulsion of ionic surfactants and (2) reducing the amount of monomeric ionic surfactant available for interaction with the enzyme (i.e., by lowering the critical micelle concentration). Additionally, our results illustrate that there is a poor correlation between thermodynamic stability in buffer (DeltaG(unfolding)) and resistance to surfactant-induced unfolding.     

Low-cost equilibrium unfolding of heme proteins using 2 μl samples

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure–function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b₅, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed.     

Unfolding study of a trimeric membrane protein AcrB

The folding of a multi‐domain trimeric α‐helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two‐state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter‐subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate‐limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrB_Δloop, from the SDS unfolded state. The CD spectrum of the refolded AcrB_Δloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re‐association of the trimer might be the limiting factor to obtain folded wild‐type AcrB.     

Thermal stability of proteins analyzed by temperature scanning pH-stat titration

Based on the fact that pH changes occur during the thermal unfolding of a protein, a pH-stat titrimetric procedure is described for the analysis of thermal stability. In all cases the agreement with other stability measurements was good, including a correlation with activity loss in enzymes. A model for the titration curves, assuming first-order denaturation kinetics, linear temperature increase, and validity of the Arrhenius equation, has been proposed and analyzed. Thus, thermodynamic constants can be calculated from tritration curves, or transition temperatures estimated if the Arrhenius constants are known. The equipment consists of a pH-stat, a programmable heating unit, and a temperature measuring/recording system. Analysis can be done quickly and on partially purified solutions, provided the buffer capacity is low, using about 20 mg protein/10 ml sample. The effects of pH, Ca²⁺ ions, substrate, chemical modification, etc., on thermal stability are conveniently analyzed up to about 90°C.     

LpxA: A natural nanotube

UDP‐N‐acetylglucosamine 3‐O‐acyltransferase is a protein with a left‐handed parallel β‐helix, which is a natural nanotube. They are associated with unusual high stability. To identify the reason behind the structural stability of β‐helical nanotubular structure, we have performed a total of 4 μs molecular dynamics simulations of the protein in implicit solvent at four different temperatures and monitored the unfolding pathway. The correlation in movement between different regions of the nanotubular structure has been identified from the dynamical cross‐correlation map and contribution of some specific residues towards unfolding transition has been identified by principal component analysis. Difference in stability of the three loop regions has also been characterized. Construction of the unfolding conformational energy landscape identifies the probable intermediates that can appear in the unfolding pathway of the protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 845–853, 2010.     

Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman’s assay

Ellman’s assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1 M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman’s assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman’s method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman’s method into two successive steps that may be convenient for some applications.     

Comparison of fluorescence and light scattering based methods to assess formation and stability of protein-protein complexes

Thermal shift methods such as differential scanning fluorimetry and differential static light scattering are widely used to identify stabilizing conditions for proteins that might promote crystallization. Here we report a comparison of the two methods when applied to optimization of buffer conditions for protein-protein complexes. Most of the protein complexes under study were amenable to analysis using these two techniques. Protein complexes behave towards thermal denaturation in a manner similar to single proteins, showing a more or less sharp transition consistent with a two-state model of unfolding. A comparison of the melting and aggregation temperatures for single components and the reconstituted complexes can provide additional evidence for complex formation and can be used to identify buffer conditions in which protein-protein complex formation is favored.     

Development and Application of a High Throughput Protein Unfolding Kinetic Assay

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses.     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.