Mechanical Deformation Accelerates Protein Ageing

A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week‐long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.     

Water‐in‐Oil Micro‐Emulsion Enhances the Secondary Structure of a Protein by Confinement

A scheme is presented in which an organic solvent environment in combination with surfactants is used to confine a natively unfolded protein inside an inverse microemulsion droplet. This type of confinement allows a study that provides unique insight into the dynamic structure of an unfolded, flexible protein which is still solvated and thus under near‐physiological conditions. In a model system, the protein osteopontin (OPN) is used. It is a highly phosphorylated glycoprotein that is expressed in a wide range of cells and tissues for which limited structural analysis exists due to the high degree of flexibility and large number of post‐translational modifications. OPN is implicated in tissue functions, such as inflammation and mineralisation. It also has a key function in tumour metastasis and progression. Circular dichroism measurements show that confinement enhances the secondary structural features of the protein. Small‐angle X‐ray scattering and dynamic light scattering show that OPN changes from being a flexible protein in aqueous solution to adopting a less flexible and more compact structure inside the microemulsion droplets. This novel approach for confining proteins while they are still hydrated may aid in studying the structure of a wide range of natively unfolded proteins.     

Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra

Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.     

Protein structure along the order-disorder continuum

Thermal fluctuations cause proteins to adopt an ensemble of conformations wherein the relative stability of the different ensemble members is determined by the topography of the underlying energy landscape. "Folded" proteins have relatively homogeneous ensembles, while "unfolded" proteins have heterogeneous ensembles. Hence, the labels "folded" and "unfolded" represent attempts to provide a qualitative characterization of the extent of structural heterogeneity within the underlying ensemble. In this work, we introduce an information-theoretic order parameter to quantify this conformational heterogeneity. We demonstrate that this order parameter can be estimated in a straightforward manner from an ensemble and is applicable to both unfolded and folded proteins. In addition, a simple formula for approximating the order parameter directly from crystallographic B factors is presented. By applying these metrics to a large sample of proteins, we show that proteins span the full range of the order-disorder axis.     

Design of a hyperstable protein by rational consideration of unfolded state interactions

Stabilization of proteins is a long-sought objective. Targeting the unfolded state interactions of a protein is not a method used for this purpose, although many proteins are known to contain such interactions. The N-terminal domain of ribosomal protein L9 (NTL9) has a lysine residue at position 12, which makes strong non-native interactions in the unfolded state. Substitution of a d-alanine for G34 in NTL9 is known to stabilize the protein by reducing the entropy of the unfolded state. Here we combine these two mutations to design a hyperstable protein. The structure of the variant is the same as that of wild-type as judged by 2D NMR. The variant is hyperstable as judged by denaturation experiments, where complete thermal unfolding of the protein does not occur in native buffer.     

Statistical theory for protein ensembles with designed energy landscapes

Combinatorial protein libraries provide a promising route to investigate the determinants and features of protein folding and to identify novel folding amino acid sequences. A library of sequences based on a pool of different monomer types are screened for folding molecules, consistent with a particular foldability criterion. The number of sequences grows exponentially with the length of the polymer, making both experimental and computational tabulations of sequences infeasible. Herein a statistical theory is extended to specify the properties of sequences having particular values of global energetic quantities that specify their energy landscape. The theory yields the site-specific monomer probabilities. A foldability criterion is derived that characterizes the properties of sequences by quantifying the energetic separation of the target state from low-energy states in the unfolded ensemble and the fluctuations of the energies in the unfolded state ensemble. For a simple lattice model of proteins, excellent agreement is observed between the theory and the results of exact enumeration. The theory may be used to provide a quantitative framework for the design and interpretation of combinatorial experiments.     

The pentapeptide GGAGG has PII conformation

Most of what we know about proteins reflects their native folded structure. Much less is understood about the structure of unfolded proteins, which tends to be referred to as "random coil", lacking extended alpha-helix or beta-strand structure. Recent work suggests that unfolded proteins might adopt significant population of PII structure, an extended left-handed helix found in collagen and proline-rich peptides. A series of short peptides AcGGXGGNH2 has been adopted as a model for studying unfolded protein structure because of the minimal steric effect imposed by flanking glycines. Peptide AcGGAGGNH2 makes possible a host-guest conformation analysis of the middle residue alanine. NMR experiments reveal that the Phi and Psi dihedral angles of the central alanine are -73 degrees and 125 degrees , respectively, placing the alanine in the PII region of the Ramachandran plot. Circular dichroism shows a typical PII spectrum with a strong negative absorbance at 190 nm. Temperature experiments show the alanine structure shifts to increasing beta-strand at high temperature. Because the alanine side chain most closely represents unsubstituted peptide backbone, these results have significant implications for the conformational entropy of unfolded polypeptide chains.     

Understanding cold denaturation: the case study of Yfh1

All globular proteins undergo transitions from their native to unfolded states if exposed either to cold or to heat perturbation. While the heat-induced transition is well described for a large number of proteins, in media compatible with natural environments, the limited number of examples of cold denatured states concern proteins artificially destabilized, for instance, by the presence of denaturants, ad hoc point mutations, or both. Here, we provide a characterization of the low temperature unfolded state of Yfh1, a natural protein that undergoes cold denaturation around water freezing temperature, in the absence of any denaturant. By achieving nearly full assignment of the NMR spectrum, we show that at -1 °C, Yfh1 has all the features of an unfolded protein, although retaining some local, residual secondary structure. The effect is not uniform along the sequence and does not merely reflect the secondary structural features of the folded species. The N-terminus seems to be dynamically more flexible, although retaining some nascent helix character. Interestingly, this region is the one containing functionally important hot-spots. The β-sheet region and the C-terminal helix are completely unfolded, although experiencing some conformational exchange, partly due to the presence of several prolines. Ours is the first step toward a full characterization of the low temperature unfolded state of a natural protein, reached without the aid of any destabilizing agent. We discuss the implications of our findings for understanding cold denatured states.     

Sequence-dependent correction of random coil NMR chemical shifts.

Random coil chemical shifts are commonly used to detect secondary structure elements in proteins in chemical shift index calculations. While this technique is very reliable for folded proteins, application to unfolded proteins reveals significant deviations from measured random coil shifts for certain nuclei. While some of these deviations can be ascribed to residual structure in the unfolded protein, others are clearly caused by local sequence effects. In particular, the amide nitrogen, amide proton, and carbonyl carbon chemical shifts are highly sensitive to the local amino acid sequence. We present a detailed, quantitative analysis of the effect of the 20 naturally occurring amino acids on the random coil shifts of (15)N(H), (1)H(N), and (13)CO resonances of neighboring residues, utilizing complete resonance assignments for a set of five-residue peptides Ac-G-G-X-G-G-NH(2). The work includes a validation of the concepts used to derive sequence-dependent correction factors for random coil chemical shifts, and a comprehensive tabulation of sequence-dependent correction factors that can be applied for amino acids up to two residues from a given position. This new set of correction factors will have important applications to folded proteins as well as to short, unstructured peptides and unfolded proteins.     

A Maleimide‐functionalized Tetraphenylethene for Measuring and Imaging Unfolded Proteins in Cells

Collapse of the protein homeostasis (proteostasis) can lead to accumulation and aggregation of unfolded proteins, which has been found to associate with a number of disease conditions including neurodegenerative diseases, diabetes and inflammation. Here we report a maleimide‐functionalized tetraphenylethene (TPE)‐derivatized fluorescent dye, TPE‐NMI, which shows fluorescence turn‐on property upon reacting with unfolded proteins in vitro and in live cells under proteostatic stress conditions. The level of unfolded proteins can be measured by flow cytometry and visualized with confocal microscopy.     

Directing the secondary structure of polypeptides at will: from helices to amyloids and back again?

An ageing society faces an increasing number of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Creutzfeld-Jacob disease. The deposition of amyloid fibrils is a pathogenic factor causing the destruction of neuronal tissue. Amyloid-forming proteins are mainly alpha-helical in their native conformation, but undergo an alpha-helix to beta-strand conversion before or during fibril formation. Partially unfolded or misfolded beta-sheet fragments are discussed as direct precursors of amyloids. To potentially cure neurodegenerative diseases we need to understand the complex folding mechanisms that shift the equilibrium from the functional to the pathological isoform of the proteins involved. This paper describes a novel approach that allows us to study the interplay between peptide primary structure and environmental conditions for peptide and protein folding in its whole complexity on a molecular level. This de novo designed peptide system may achieve selective inhibition of fibril formation.     

Molecular simulation of protein dynamics in nanopores. I. Stability and folding

Discontinuous molecular dynamics simulations, together with the protein intermediate resolution model, an intermediate-resolution model of proteins, are used to carry out several microsecond-long simulations and study folding transition and stability of alpha-de novo-designed proteins in slit nanopores. Both attractive and repulsive interaction potentials between the proteins and the pore walls are considered. Near the folding temperature T(f) and in the presence of the attractive potential, the proteins undergo a repeating sequence of folding/partially folding/unfolding transitions, with T(f) decreasing with decreasing pore sizes. The unfolded states may even be completely adsorbed on the pore's walls with a negative potential energy. In such pores the energetic effects dominate the entropic effects. As a result, the unfolded state is stabilized, with a folding temperature T(f) which is lower than its value in the bulk and that, compared with the bulk, the folding rate decreases. The opposite is true in the presence of a repulsive interaction potential between the proteins and the walls. Moreover, for short proteins in very tight pores with attractive walls, there exists an unfolded state with only one alpha-helical hydrogen bond and an energy nearly equal to that of the folded state. The proteins have, however, high entropies, implying that they cannot fold onto their native structure, whereas in the presence of repulsive walls the proteins do attain their native structure. There is a pronounced asymmetry between the two termini of the protein with respect to their interaction with the pore walls. The effect of a variety of factors, including the pore size and the proteins' length, as well as the temperature, is studied in detail.     

Dynamics of unfolded protein transport through an aerolysin pore

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.     

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.     

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.     

Predicting melting temperature directly from protein sequences

Proteins of both hyperthermophilic and mesophilic microorganisms generally constitute from the same 20 amino acids; however, the extent of thermal tolerance of any given protein is an inherent property of its amino acid sequence. The present study is the first to report a rapid method for predicting Tm (melting temperature), the temperature at which 50% of the protein is unfolded, directly from protein sequences (the Tm Index program is available at http://tm.life.nthu.edu.tw/). We examined 75 complete microbial genomes using the Tm Index, and the analysis clearly differentiated hyperthermophilic from mesophilic microorganisms on this global genomic basis. These results are consistent with the previous hypothesis that hyperthermophiles express a greater number of high Tm proteins compared with mesophiles. The Tm Index will be valuable for modifying existing proteins (enzymes, protein drugs and vaccines) or designing novel proteins having a desired melting temperature.     

Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding

Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.     

Inclusion complexes of proteins: Interaction of cyclodextrins with peptides containing aromatic amino acids studied by competitive spectrophotometry

The stability constants were measured of inclusion complexes formed from aromatic amino acids and their oligopeptides with - and-cyclodextrin, hydroxypropyl-cyclodextrin, and partially methylated-cyclodextrin. The method of competitive spectrophotometry withp-nitrophenol as a competing reagent was used, and measurements were made at pH 7.4-Cyclodextrin formed complexes of higher stability than the other hosts. The stability of complexes of oligopeptides containing L-phenylalanine was invariably higher than that of L-phenylalanine itself. A model for interaction of proteins with cyclodextrins is proposed, in which the most stable complexes are formed when the native functional form of proteins is unfolded and the nonpolar residues that are buried inside the structure are exposed to water. The complexation of the unfolded structure favors its formation; thus thermal denaturation of proteins is easier in the presence of cyclodextrins. On the other hand, this complexation prevents the intermolecular association of unfolded structures by noncovalent hydrophobic bonding between the exposed nonpolar residues; furthermore, the unfolded complexed forms may revert to the native functional form. This prevention of intermolecular association may explain the stabilizing effect of cyclodextrins on solutions of proteins: a return to the native form is achieved more easily from the complexed, unfolded form than from the unfolded, aggregated forms.Dedicated to Professor József Szejtli.