Misfolding of Luciferase at the Single‐Molecule Level

The folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding.     

Evidence of complete hydrophobic coating of bombesin by trifluoroethanol in aqueous solution: an NMR spectroscopic and molecular dynamics study

Bombesin is a tetradecapeptide that possesses a random coil structure in pure water. In the presence of 30 % (v/v) 2,2,2-trifluoroethanol (TFE), it adopts a partial helical conformation involving the C-terminal amino acids 6-14. This conformational change, known as the TFE effect, is studied here in terms of the solvation state of the peptide at different TFE concentrations by means of intermolecular homo- and heteronuclear NOE measurements. When an aqueous solution of bombesin is titrated with TFE, a continual decrease in the water/peptide interactions and a concomitant increase in the TFE/peptide interactions is observed, and at 30 % (v/v) TFE no homonuclear NOEs between water and the peptide can be detected. The conformational transition of the bombesin molecule is thus accompanied by a complete surface covering with TFE. A parallel molecular dynamics (MD) study of the peptide in aqueous solution with the single-point charge (SPC) water model and in a 30 % (v/v) TFE/water mixture with a recently developed TFE model has also been performed. The 10 ns simulations were in agreement with the experimental data. The calculations indicate stabilisation of the alpha-helix in the H(2)O/TFE mixture, in contrast to the situation in pure water, and clustering of the TFE molecules around the peptide.     

Molecular simulation of surfactant-assisted protein refolding

Protein refolding to its native state in vitro is a challenging problem in biotechnology, i.e., in the biomedical, pharmaceutical, and food industry. Protein aggregation and misfolding usually inhibit the recovery of proteins with their native states. These problems can be partially solved by adding a surfactant into a suitable solution environment. However, the process of this surfactant-assisted protein refolding is not well understood. In this paper, we wish to report on the first-ever simulations of surfactant-assisted protein refolding. For these studies, we defined a simple model for the protein and the surfactant and investigated how a surfactant affected the folding behavior of a two-dimensional lattice protein molecule. The model protein and model surfactant were chosen such that we could capture the important features of the folding process and the interaction between the protein and the surfactant, namely, the hydrophobic interaction. It was shown that, in the absence of surfactants, a protein in an "energy trap" conformation, i.e., a local energy minima, could not fold into the native form, which was characterized by a global energy minimum. The addition of surfactants created folding pathways via the formation of protein-surfactant complexes and thus enabled the conformations that fell into energy trap states to escape from these traps and to form the native proteins. The simulation results also showed that it was necessary to match the hydrophobicity of surfactant to the concentration of denaturant, which was added to control the folding or unfolding of a protein. The surfactants with different hydrophobicity had their own concentration range on assisting protein refolding. All of these simulations agreed well with experimental results reported elsewhere, indicating both the validity of the simulations presented here and the potential application of the simulations for the design of a surfactant on assisting protein refolding.     

Sulfonated molecules that bind a partially structured species of beta2-microglobulin also influence refolding and fibrillogenesis

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.     

Single Molecule Characterization of Amyloid Oligomers

The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.     

Protein refolding assisted by periodic mesoporous organosilicas

Herein we report a new strategy for protein refolding by taking advantage of the unique surface and pore characteristics of ethylene-bridged periodic mesoporous organosilica (PMO), which can effectively entrap unfolded proteins and assist refolding by controlled release into the refolding buffer. Hen egg white lysozyme was used as a model protein to demonstrate the new method of protein refolding. Through loading of denatured proteins inside uniform mesoporous channels tailored to accommodate individual protein, protein aggregation was minimized, and the folding rate was increased. Poly(ethyleneglycol) (PEG)-triggered continuous release of entrapped denatured lysozyme allowed high-yield refolding with high cumulative protein concentrations. The new method enhances the oxidative refolding of lysozyme (e.g., over 80% refolding yield at about 0.6 mg/mL).     

Infinite Assembly of Folded Proteins in Evolution,Disease, and Engineering

Mutations and changes in a protein's environment are well known for their potential to induce misfolding and aggregation, including amyloid formation. Alternatively, such perturbations can trigger new interactions that lead to the polymerization of folded proteins. In contrast to aggregation, this process does not require misfolding and, to highlight this difference, we refer to it as agglomeration. This term encompasses the amorphous assembly of folded proteins as well as the polymerization in one, two, or three dimensions. We stress the remarkable potential of symmetric homo‐oligomers to agglomerate even by single surface point mutations, and we review the double‐edged nature of this potential: how aberrant assemblies resulting from agglomeration can lead to disease, but also how agglomeration can serve in cellular adaptation and be exploited for the rational design of novel biomaterials.     

The effects of denaturants on protein conformation and behavior at air/solution interface

In this study, we discuss the interfacial behavior of five proteins with different conformational character, and each is investigated in native and denatured states. The protein molecules are layered and spread onto the air/solution interfaces to form protein monolayer. The surface pressure-time (Pi(t)) and surface pressure-area per molecule (Pi-A) isotherms were measured by using the Langmuir-Blodgett (LB) balance consisted of a Nima trough system. The differences between monolayered protein's behaviors at air/solution interface indicate that denaturants, such as urea, guanidinium chloride and dithiothreitol, have different effects on conformational changes of proteins. Additionally, the interfacial behavior of the proteins in our study provides a fundamental profile about the protein structural stability and implies industrial applications in protein refolding process.     

Structure and folding of glucagon-like peptide-1-(7–36)-amide in aqueous trifluoroethanol studied by NMR spectroscopy

The conformational changes of free, monomeric glucagon-like peptide-1-(7–36)-amide (GLP-1) in aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from 292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil (in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more stable than the N-terminal part. Copyright © 2001 John Wiley & Sons, Ltd.     

KOH catalysed preparation of activated carbon aerogels for dye adsorption

Surfactants prevent the irreversible aggregation of partially refolded proteins, and they are also known to assist in protein refolding. A novel approach to protein refolding that utilizes a pair of low molecular weight folding assistants, a detergent and cyclodextrin, was proposed by Rozema and Gellman (D. Rozema, S.H. Gellman, J. Am. Chem. Soc. 117 (1995) 2373). We report the refolding of bovine serum albumin (BSA) assisted by these artificial chaperones, utilizing gemini surfactants for the first time. A combination of cationic gemini surfactants, bis(cetyldimethylammonium)pentane dibromide (C(16)H(33)(CH(3))(2)N(+)-(CH(2))(5)-N(+)(CH(3))(2)C(16)H(33)·2Br(-) designated as G5 and bis(cetyldimethylammonium)hexane dibromide (C(16)H(33)(CH(3))(2)N(+)-(CH(2))(6)-N(+)(CH(3))(2)C(16)H(33)·2Br(-) designated as G6 and cyclodextrins, was used to refold guanidinium chloride (GdCl) denatured BSA in the artificial chaperone assisted two step method. The single chain cationic surfactant cetyltrimethylammonium bromide (CTAB) was used for comparative studies. The studies were carried out in an aqueous medium at pH 7.0 using circular dichroism, dynamic light scattering and ANS binding studies. The denatured BSA was found to get refolded by very small concentrations of gemini surfactant at which the single chain counterpart was found to be ineffective. Different from the single chain surfactant, the gemini surfactants exhibit much stronger electrostatic and hydrophobic interactions with the protein and are thus effective at much lower concentrations. Based on the present study it is expected that gemini surfactants may prove useful in the protein refolding operations and may thus be effectively employed to circumvent the problem of misfolding and aggregation.     

The Important Roles of Water in Protein Folding: an Approach by Single Molecule Force Spectroscopy

The single-chain elasticity of a completely unfolded protein ((I27)8,modules of human cardiac titin) is studied in different liquid environments by the atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS).The experimental results show that there is a clear deviation between the force curves obtained in the aqueous and nonaqueous environments.Such a deviation can be attributed to the additional energy consumed by the rearrangement of the bound water molecules around the chain of the completely unfolded (I27)8 chain upon stretching in aqueous solution,which is very similar to the partial dehydration process from a denatured/unfolded to a native/folded protein.Through the analysis of the free energy changes involved in protein folding,we conclude that it is due to the weak disturbance of water molecules and the special backbone structures of proteins that the self-assembly of proteins can be achieved in physiological conditions.We speculate that water is likely to be an important criterion for the selection of self-assembling macromolecules in the prebiotic chemical evolution.     

Topography of the free-energy landscape probed via mechanical unfolding of proteins

Single-molecule experiments in which proteins are unfolded by applying mechanical stretching forces generally force unfolding to proceed along a reaction coordinate that is different from that in chemical or thermal denaturation. Here we simulate the mechanical unfolding and refolding of a minimalist off-lattice model of the protein ubiquitin to explore in detail the slice of the multidimensional free-energy landscape that is accessible via mechanical pulling experiments. We find that while the free-energy profile along typical "chemical" reaction coordinates may exhibit two minima, corresponding to the native and denatured states, the free energy G(z) is typically a monotonic function of the mechanical coordinate z equal to the protein extension. Application of a stretching force along z tilts the free-energy landscape resulting in a bistable (or multistable) free energy G(z)-fz probed in mechanical unfolding experiments. We construct a two-dimensional free-energy surface as a function of both chemical and mechanical reaction coordinates and examine the coupling between the two. We further study the refolding trajectories after the protein has been prestretched by a large force, as well as the mechanical unfolding trajectories in the presence of a large stretching force. We demonstrate that the stretching forces required to destabilize the native state thermodynamically are larger than those expected on the basis of previous experimental estimates of G(z). This finding is consistent with the recent experimental studies, indicating that proteins may refold even in the presence of a substantial stretching force. Finally, we show that for certain temperatures the free energy of a polyprotein chain consisting of multiple domains is a linear function of the chain extension. We propose that the recently observed "slow phase" in the refolding of proteins under mechanical tension may be viewed as downhill diffusion in such a linear potential.     

Aggregation of amyloid Aβ(1–40) peptide in perdeuterated 2,2,2‐trifluoroethanol caused by ultrasound sonication

Ultrasound sonication of protein and peptide solutions is routinely used in biochemical, biophysical, pharmaceutical and medical sciences to facilitate and accelerate dissolution of macromolecules in both aqueous and organic solvents. However, the impact of ultrasound waves on folding/unfolding of treated proteins, in particular, on aggregation kinetics of amyloidogenic peptides and proteins is not understood. In this work, effects of ultrasound sonication on the misfolding and aggregation behavior of the Alzheimer's Aβ_(1–40)‐peptide is studied by pulsed‐field gradient (PFG) spin–echo diffusion NMR and UV circular dichroism (CD) spectroscopy. Upon simple dissolution of Aβ_(1–40) in perdeuterated trifluoroethanol, CF₃‐CD₂‐OD (TFE‐d₃), the peptide is present in the solution as a stable monomer adopting α‐helical secondary structural motifs. The self‐diffusion coefficient of Aβ_(1–40) monomers in TFE‐d₃ was measured as 1.35 × 10^?10 m² s^?1, reflecting its monomeric character. However, upon ultrasonic sonication for less than 5 min, considerable populations of Aβ molecules (ca 40%) form large aggregates as reflected in diffusion coefficients smaller than 4.0 × 10^?13 m² s^?1. Sonication for longer times (up to 40 min in total) effectively reduces the fraction of these aggregates in ¹H PFG NMR spectra to ca 25%. Additionally, absorption below 230 nm increased significantly upon sonication treatment, an observation, which also clearly confirms the ongoing aggregation process of Aβ_(1–40) in TFE‐d₃. Surprisingly, upon ultrasound sonication only small changes in the peptide secondary structure were detected by CD: the peptide molecules mainly adopt α‐helical motifs in both monomers and aggregates formed upon sonication. Copyright © 2010 John Wiley & Sons, Ltd.     

New force replica exchange method and protein folding pathways probed by force-clamp technique

We have developed a new extended replica exchange method to study thermodynamics of a system in the presence of external force. Our idea is based on the exchange between different force replicas to accelerate the equilibrium process. This new approach was applied to obtain the force-temperature phase diagram and other thermodynamical quantities of the three-domain ubiquitin. Using the C(alpha)-Go model and the Langevin dynamics, we have shown that the refolding pathways of single ubiquitin depend on which terminus is fixed. If the N end is fixed then the folding pathways are different compared to the case when both termini are free, but fixing the C terminal does not change them. Surprisingly, we have found that the anchoring terminal does not affect the pathways of individual secondary structures of three-domain ubiquitin, indicating the important role of the multidomain construction. Therefore, force-clamp experiments, in which one end of a protein is kept fixed, can probe the refolding pathways of a single free-end ubiquitin if one uses either the polyubiquitin or a single domain with the C terminus anchored. However, it is shown that anchoring one end does not affect refolding pathways of the titin domain I27, and the force-clamp spectroscopy is always capable to predict folding sequencing of this protein. We have obtained the reasonable estimate for unfolding barrier of ubiquitin, using the microscopic theory for the dependence of unfolding time on the external force. The linkage between residue Lys48 and the C terminal of ubiquitin is found to have the dramatic effect on the location of the transition state along the end-to-end distance reaction coordinate, but the multidomain construction leaves the transition state almost unchanged. We have found that the maximum force in the force-extension profile from constant velocity force pulling simulations depends on temperature nonlinearly. However, for some narrow temperature interval this dependence becomes linear, as have been observed in recent experiments.     

Detection of the equilibrium folding intermediate of beta-lactoglobulin in the presence of trifluoroethanol by mass spectrometry

Nano-electrospray ionization mass spectrometry (nano-ESI-MS) was used to monitor the effect of trifluoroethanol (TFE) on the conformational properties of beta-lactoglobulin (BLG). TFE stabilizes protein secondary structure, particularly alpha-helices. However, it also acts as a denaturant above critical concentrations. In the case of BLG, TFE at low concentrations is known to induce formation of an equilibrium intermediate that contains non-native helical structure. Such an intermediate is thought to form also under physiological conditions, playing a role in BLG folding in vivo by preventing aggregation. This well-characterized system was chosen in order to test species distributions obtained by nano-ESI-MS. BLG spectra at increasing concentrations of TFE at pH 2 indicate transient accumulation of a conformer whose charge-state distribution (CSD) falls between that of the native and that of the denatured protein, indicating that the TFE-induced, partially folded form can be selectively monitored by this technique. The condition of its maximum accumulation corresponds to 16% TFE, in excellent agreement with results from solution experiments. In contrast, titrations with methanol or acetonitrile (ACN) reveal apparent two-state transitions from native to fully unfolded BLG. At 10% TFE, the protein appears to be still fully folded at room temperature but, if unfolding is elicited by the combination with other denaturing agents, e.g. heat or low concentrations of ACN, it proceeds via formation of the intermediate. Thus, TFE can also induce formation of the BLG intermediate in synergism with generic denaturing agents. This study indicates good agreement between ESI-MS and other biophysical methods monitoring protein conformational transitions in the presence of TFE.     

The mechanism of GroEL/GroES folding/refolding of protein substrates revisited

The thermodynamics and kinetics of zinc-cytochrome c (ZnCyt c) interactions with Escherichia coli molecular chaperone GroEL (Chaperonin 60; Cpn60) are described. Zinc(II)-porphyrin represents a flexible fluorescent probe for thermodynamic complex formation between GroEL and ZnCyt c, as well as for stopped-flow fluorescence kinetic experiments. Data suggests that GroEL and GroEL/GroES-assisted refolding of unfolded ZnCyt c takes place by a mechanism that is quite close to the Anfinsen Cage hypothesis for molecular chaperone activity. However, even in the presence of ATP, GroEL/GroES-assisted refolding of ZnCyt c takes place at approximately half the rate of refolding of ZnCyt c alone. On the other hand, there is little evidence for refolding behaviour consistent with the Iterative Annealing hypothesis. This includes a complete lack of GroEL or GroEL/GroES-assisted enhancement of refolding rate constant k(2) associated with the unfolding of a putative misfolded state I (Zn) on the pathway to the native state. Reviewing our data in the light of data from other laboratories, we observe that all forward rate enhancements or reductions could be accounted for in terms of thermodynamic coupling (adjusting positions of refolding equilibria) due to binding interactions between GroEL and unfolded protein substrates, driven by thermodynamic considerations. Therefore, we propose that passive kinetic partitioning should be considered the core mechanism of the GroEL/GroES molecular chaperone machinery, wherein the core function is to bind unfolded protein substrates leading to a blockade of aggregation pathways and to increases in molecular flux through productive folding pathway(s).     

Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography

α-Fetoprotein (AFP) is a prospective biopharmaceutical candidate currently undergoing advanced-stage clinical trials for autoimmune indications. The high AFP expression yields in the form of inclusion bodies in Escherichia coli renders the inclusion body route potentially advantageous for process scale commercial manufacture, if high-throughput refolding can be achieved. This study reports the successful development of an ‘anion-exchange chromatography’-based refolding process for recombinant human AFP (rhAFP), which carries the challenges of contaminant spectrum and molecule complexity. rhAFP was readily refolded on-column at rhAFP concentrations unachievable with dilution refolding due to viscosity and solubility constraints. DEAE-FF functioned as a refolding enhancer to achieve rhAFP refolding yield of 28% and product purity of 95% in 3 h, at 1 mg/ml protein refolding concentration. Optimization of both refolding and chromatography column operation parameters (i.e. resin chemistry, column geometry, redox potential and feed conditioning) significantly improved rhAFP refolding efficiency. Compared to dilution refolding, on-column rhAFP refolding productivity was 9-fold higher, while that of off-column refolding was more than an order of magnitude higher. Successful demonstration that a simple anion-exchange column can, in a single step, readily refold and purify semi-crude rhAFP comprising 16 disulfide bonds, will certainly extend the application of column refolding to a myriad of complex industrial inclusion body proteins.     

Direct Identification of Protein–Protein Interactions by Single‐Molecule Force Spectroscopy

Single‐molecule force spectroscopy based on atomic force microscopy (AFM‐SMFS) has allowed the measurement of the intermolecular forces involved in protein‐protein interactions at the molecular level. While intramolecular interactions are routinely identified directly by the use of polyprotein fingerprinting, there is a lack of a general method to directly identify single‐molecule intermolecular unbinding events. Here, we have developed an internally controlled strategy to measure protein–protein interactions by AFM‐SMFS that allows the direct identification of dissociation force peaks while ensuring single‐molecule conditions. Single‐molecule identification is assured by polyprotein fingerprinting while the intermolecular interaction is reported by a characteristic increase in contour length released after bond rupture. The latter is due to the exposure to force of a third protein that covalently connects the interacting pair. We demonstrate this strategy with a cohesin–dockerin interaction.     

MMP-12 蛋白包涵体复性时折叠状态核磁共振图谱与荧光光谱研究

MMP-12 是癌症治疗药物靶标. 为了更好研制新药, 需要大量制备MMP-12, 但MMP-12 在大肠杆菌中以包涵体形式表达. 因此如何优化蛋白复性过程是大量获取MMP-12 蛋白的关键. 采用核磁共振、稳态荧光法、外源性ANS (8-anilinol-naphthalenesulfonic acid)荧光探针三种方法监控MMP-12 变性蛋白的再折叠过程, 以探究其复性折叠机制. 研究发现MMP-12 再折叠中点值与对应的尿素浓度几乎相等(C_m≈4, mid-point of transition). 不同尿素浓度中MMP-12 的二维¹H-¹⁵N HSQC (heteronuclear single quantum correlation)谱图显示, 尿素浓度从4 mol/L 降低到3 mol/L 是MMP-12 蛋白复性折叠的关键步骤. 据此我们将MMP-12 蛋白复性从常规的梯度透析复性方法改进成等容透析复性法(即确保尿素从4 mol/L 到3 mol/L 的浓度变化缓慢), 实现复性收率提高一倍.     

Quantitative determination of uridine in rabbit plasma and urine by liquid chromatography coupled to a tandem mass spectrometry

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits.