Gas‐phase behavior of noncovalent transmembrane segment complexes

Specific helix oligomerization between transmembrane segments (TMSs) is often promoted by motifs like GxxxG. Disruption of this motif in the transmembrane segments of vesicular stomatitis virus G‐protein and of glycophorin A results in a reduced dimerization level studied by in vivo systems like ToxR. This paper reports the influence of sequence motifs like GxxxG in solution and the gas phase. The transmembrane segments may behave differently in the gas and liquid phase, because of the absence of surrounding solvent molecules in the gas phase. Comparison of experiments depending on peptide properties performed in the gas and liquid phase discloses that the peptides retain ‘some memory’ of their liquid‐phase structure in the gas phase. A direct correlation has been found between helicity in solution as determined by circular dichroism and dimerization in the gas phase monitored by electrospray mass spectrometry. These results show that a proper folding in solution is required for oligomerization. On the other hand, sequence‐specific oligomerization depending on the GxxxG motif was not observed with the mass spectrometric detection. Further on, neither concentration‐dependent complex studies nor studies regarding complex stability in the gas phase – via collision‐induced dissociation (CID) – led to sequence‐specific differences. Finally, the findings show that in mass spectrometric measurements noncovalent interactions of studied TMSs is rather more dependent on the secondary structure and proper folding than on their primary structure. Copyright © 2008 John Wiley & Sons, Ltd.     

Native Biomolecules in the Gas Phase? The Case of Green Fluorescent Protein

Green fluorescent protein (GFP) was ionized by native electrospray ionization and trapped for many seconds in high vacuum, allowing fluorescence emission to be measured as a probe of its biological function, to answer the question whether GFP exists in the native form in the gas phase or not. Although a narrow charge‐state distribution, a collision cross‐section very close to that expected for correctly folded GFP, and a large stability against dissociation all support a near‐native gas‐phase structure, no fluorescence emission was observed. The loss of the native form is attributed to the absence of residual water in the gas phase, which normally stabilizes the para‐hydroxybenzylidene imidazolone chromophore of GFP.     

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins fromE. coli

The high-level expression of recombinant gene products in the gramnegative bacteriumEscherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as thecis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.     

Machine learning algorithms for predicting protein folding rates and stability of mutant proteins: comparison with statistical methods

Machine learning algorithms have wide range of applications in bioinformatics and computational biology such as prediction of protein secondary structures, solvent accessibility, binding site residues in protein complexes, protein folding rates, stability of mutant proteins, and discrimination of proteins based on their structure and function. In this work, we focus on two aspects of predictions: (i) protein folding rates and (ii) stability of proteins upon mutations. We briefly introduce the concepts of protein folding rates and stability along with available databases, features for prediction methods and measures for prediction performance. Subsequently, the development of structure based parameters and their relationship with protein folding rates will be outlined. The structure based parameters are helpful to understand the physical basis for protein folding and stability. Further, basic principles of major machine learning techniques will be mentioned and their applications for predicting protein folding rates and stability of mutant proteins will be illustrated. The machine learning techniques could achieve the highest accuracy of predicting protein folding rates and stability. In essence, statistical methods and machine learning algorithms are complimenting each other for understanding and predicting protein folding rates and the stability of protein mutants. The available online resources on protein folding rates and stability will be listed.     

Flexible Folding: Disulfide-Containing Peptides and Proteins Choose the Pathway Depending on the Environments

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.     

固体表面特征对脲变α-糜蛋白酶折叠的贡献

以脲变α-糜蛋白酶(α-Chy)为模型蛋白, 用蛋白折叠液相色谱法研究了该蛋白在7种不同固体表面上的折叠及其在折叠过程中形成的中间体, 选用疏水相互作用色谱(HPHIC)固定相为吸附剂, 在动态条件下着重研究了疏水色谱固定相TSK和PEG-600表面对脲变α-Chy复性效率的贡献. 用基质辅助激光解吸附离子化飞行时间质谱对3.0 mol•L^－1脲变α-Chy, 在经 HPHIC柱复性并同时分离的收集组分进行确认后, 仅有一种稳定的脲变α-Chy折叠中间体. 发现PEG-600固定相表面较TSK固定相对α-Chy复性效果好. 证实了疏水性强度及固体表面配基的结构对蛋白折叠起着关键性的作用.     

Quantification of solvent water and hydration dynamic of thermo‐sensitive microgel by dielectric spectroscopy

The hydration of natural or synthetic macromolecules is of fundamental importance in our understanding of their structure and stability. Quantification of hydration water can promote the understanding to many complex biological mechanisms such as protein folding, as well as the dynamics and conformation of polymers. An approach to quantification of solvent water was developed by dielectric spectroscopy. Dielectric behaviors of PNIPAM microgels with different crosslink density distribution were measured in the range of 0.5–40 GHz and 15–50. An obvious relaxation process caused by free and bound water was found. Dielectric parameters of free and bound water show that the crosslink density distribution does not affect the volume phase transition temperature of microgels, but significantly influence the orientation dynamics of the solvent water. We found that the three kinds of microgel can be distinguished by the dielectric parameters of the bound water. In addition, the number of water in and outside microgel during the volume phase transition process was quantitatively calculated for the first time. This study provides the possibility for the quantification of water in complex biological process. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2017 , 55, 1859–1864     

Amyloid Polymorphism in the Protein Folding and Aggregation Energy Landscape

Protein folding involves a large number of steps and conformations in which the folding protein samples different thermodynamic states characterized by local minima. Kinetically trapped on‐ or off‐pathway intermediates are metastable folding intermediates towards the lowest absolute energy minima, which have been postulated to be the natively folded state where intramolecular interactions dominate, and the amyloid state where intermolecular interactions dominate. However, this view largely neglects the rich polymorphism found within amyloid species. We review the protein folding energy landscape in view of recent findings identifying specific transition routes among different amyloid polymorphs. Observed transitions such as twisted ribbon→crystal or helical ribbon→nanotube, and forbidden transitions such helical ribbon?crystal, are discussed and positioned within the protein folding and aggregation energy landscape. Finally, amyloid crystals are identified as the ground state of the protein folding and aggregation energy landscape.     

Intrinsically stable secondary structure elements of proteins: a comprehensive study of folding units of proteins by computation and by analysis of data determined by X-ray crystallography

Different protein architectures show strong similarities regardless of their amino acid composition: the backbone folds of the different secondary structural elements exhibit nearly identical geometries. To investigate the principles of folding and stability properties, oligopeptide models (that is, HCO-(NH-L-CHR-CO)(n)-NH(2)) have been studied. Previously, ab initio structure determinations have provided a small amount of information on the conformational building units of di- and tripeptides. A maximum of nine differently folded backbone types is available for any natural alpha-amino acid residue, with the exception of proline. All of these conformers have different relative energies. The present study compiles an ab inito database of optimized HCO-(L-Xxx)(n)-NH(2) structures, where 1相似文献   

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.     

Refolding of urea‐denatured α‐chymotrypsin by protein‐folding liquid chromatography

An approach for re‐folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α‐chymotrypsin (α‐Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α‐Chy states – urea‐denatured (U state), its folded intermediates (M state) and nature state (N state) – were studied during protein folding. Based on the test by matrix‐assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the α‐Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α‐Chy was found to be higher than that of commercial α‐Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0 m ; the highest specific bioactivity at urea concentration was 1.0 m , indicating the possibility for re‐folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded α‐Chy. When the urea concentration reached 6.0 m , the unfolded α‐Chy could not be refolded at all. Copyright © 2012 John Wiley & Sons, Ltd.     

On the Temperature–Pressure Free‐Energy Landscape of Proteins

We studied the thermodynamic stability of a small monomeric protein, staphylococcal nuclease (Snase), as a function of both temperature and pressure, and expressed it as a 3D free‐energy surface on the p,T‐plane using a second‐order Taylor expansion of the Gibbs free‐energy change ΔG upon unfolding. We took advantage of a series of different techniques (small‐angle Xray scattering, Fourier‐transform infrared spectroscopy, differential thermal analysis, pressure perturbation calorimetry and densitometry) in the evaluation of the conformation of the protein and in evaluating the changes in the thermodynamic parameters upon unfolding, such as the heat capacity, enthalpy, entropy, volume, isothermal compressibility and expansivity. The calculated results of the free‐energy landscape of the protein are in good agreement with experimental data of the p,T‐stability diagram of the protein over a temperature range from 200 to 400 K and at pressures from ambient pressure to 4000 bar. The results demonstrate that combined temperature–pressure‐dependent studies can help delineate the free‐energy landscape of proteins and hence help elucidate which features and thermodynamic parameters are essential in determining the stability of the native conformational state of proteins. The approach presented may also be used for studying other systems with so‐called re‐entrant or Tamman loop‐shaped phase diagrams.     

Engineering the protein folding landscape in gram-negative bacteria

Gram-negative bacteria, especially Escherichia coli, are often the preferred hosts for recombinant protein production because of their fast doubling times, ability to grow to high cell density, propensity for high recombinant protein titers and straightforward protein purification techniques. The utility of simple bacteria in such studies continues to improve as a result of an ever-increasing body of knowledge regarding their native protein biogenesis machinery. From translation on the ribosome to interaction with cytosolic accessory factors to transport across the inner membrane into the periplasmic space, cellular proteins interact with many different types of cellular machinery and each interaction can have a profound effect on the protein folding process. This review addresses key aspects of cellular protein folding, solubility and expression in E. coli with particular focus on the elegant biological machinery that orchestrates the transition from nascent polypeptide to folded, functional protein. Specifically highlighted are a variety of different techniques to intentionally alter the folding environment of the cell as a means to understand and engineer intracellular protein folding and stability.     

Spatial geometric arrangements of disulfide-crosslinked loops in proteins

The classification of patterns of the three-dimensional folding of a covalently crosslinked polypeptide chain can be used to introduce long-range interactions into the theoretical search for the native conformation of a protein. This classification into Spatial Geometric Arrangements of Loops (SGAL) had been proposed earlier (H. Meirovitch and H. A. Scheraga, Macromolecules 14 , 1250, 1981). It is based on the subdivision of the protein molecule into closed loops, defined by covalent crosslinks (such as disulfide bonds). Various SGAL classes correspond to the presence or absence of mutual penetration of loops, called entanglements or thrustings. A systematic and objective method is developed here to enumerate all theoretically possible SGAL's for a protein, based only on its covalent structure, i.e., the pattern of disulfide bonds or other crosslinks, regardless of whether the three-dimensional structure is known or unknown. This information can be of use in structural predictions of folding patterns. Using a modification of the method, it is also possible to determine the SGAL class to which a protein of known structure belongs. Out of 18 proteins with known three-dimensional structure and containing more than two disulfide bonds, five have a native structure with at least one entanglement or thrusting. Thus, threaded SGAL's represent a significant structural feature of native proteins. All five involve neighboring loops in the sequences. Their presence in a protein can suggest restrictions on the possible ways of folding the protein.     

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A₁ (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.     

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Ω) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.     

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.     

MALDI‐MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single‐stranded DNA‐binding protein

The Escherichia coli single‐stranded DNA binding protein (SSB) selectively binds single‐stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB?ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross‐linking, combined with high‐mass matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB?ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19 100) can be detected with standard MALDI‐MS. With chemical cross‐linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79 500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB?ssDNA complex at m/z 94 200 even in the absence of chemical cross‐linking. The stability of the SSB?ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB?ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB?ssDNA complex is how ssDNA binds to SSB, rather than the protein‐to‐DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non‐specific aggregation in the MALDI plume. Copyright © 2012 John Wiley & Sons, Ltd.     

A homology/ab initio hybrid algorithm for sampling near‐native protein conformations

One of the major challenges for protein tertiary structure prediction strategies is the quality of conformational sampling algorithms, which can effectively and readily search the protein fold space to generate near‐native conformations. In an effort to advance the field by making the best use of available homology as well as fold recognition approaches along with ab initio folding methods, we have developed Bhageerath‐H Strgen, a homology/ab initio hybrid algorithm for protein conformational sampling. The methodology is tested on the benchmark CASP9 dataset of 116 targets. In 93% of the cases, a structure with TM‐score ≥ 0.5 is generated in the pool of decoys. Further, the performance of Bhageerath‐H Strgen was seen to be efficient in comparison with different decoy generation methods. The algorithm is web enabled as Bhageerath‐H Strgen web tool which is made freely accessible for protein decoy generation ( http://www.scfbio‐iitd.res.in/software/Bhageerath‐HStrgen1.jsp ). © 2013 Wiley Periodicals, Inc.