Membrane translocation of folded proteins

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.     

The twin-arginine translocation system: a novel means of transporting folded proteins in chloroplasts and bacteria

Protein translocases have been characterised in several membrane systems and the translocation mechanisms have been shown to differ in critical respects. Nevertheless, the majority were believed to transport proteins only in a largely unfolded state, and this widespread characteristic was viewed as a likely evolutionary effort to minimise the diameter of translocation pore required. Within the last few years, however, studies on the chloroplast thylakoid membrane have revealed a novel class of protein translocase which possesses the apparently unique ability to transport fully-folded proteins across a tightly sealed energy-transducing membrane. A related system, (the twin-arginine translocation, or Tat system) has now been characterised in the Escherichia coli plasma membrane and considerations of its substrate specificity again point to its involvement in the transport of folded proteins. The emerging data suggest a critical involvement in many membranes for the biogenesis of two types of globular protein: those that are obliged to fold prior to translocation, and those that fold too tightly or rapidly for other types of protein translocase to handle.     

Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step.     

Conformations of folded proteins in restricted spaces

A new method is presented to examine the complete range of folded topologies accessible in the compact state of globular proteins. The procedure is to generate all conformations, with volume exclusion, upon a lattice in a space restricted to the individual protein's known compact conformational space. Using one lattice point per residue, we find 10(2)-10(4) possible compact conformations for the five small globular proteins studied. Subsequently, these conformations are evaluated in terms of residue-specific, pairwise contact energies that favor nonbonded, hydrophobic interactions. Native structures for the five proteins are always found within the best 2% of all conformers generated. This novel method is simple and general and can be used to determine a small group of most favorable overall arrangements for the folding of specific amino acid sequences within a restricted space.     

Alternatively folded proteins with unexpected beneficial functions

HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein-fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.     

Charge effects on folded and unfolded proteins

We develop a theory for the effects of charge on the stabilization of globular proteins. The folding process is modeled as occurring through a fictitious intermediate state along a two-part thermodynamic pathway in which the molecule (i) increases its density and then (ii) rearranges its ionic groups to the protein surface. The equilibrium for the binding of protons in salt solutions is assumed to be driven by the electrical potential due to the charge distribution, in addition to the intrinsic binding affinity and bulk proton concentration. The potential is calculated for inside and outside a porous sphere model of the protein using the Poisson-Boltzmann relation, wherein the interior dielectric constant is taken to be a linear function of the chain density. The model predicts the slope of the titration curves for native myoglobin in agreement with experiments by Breslow and Gurd (1962). From the similar experiments on the unfolded state, and from the experiments of Privalov et al. (1986) on the intrinsic viscosity of the unfolded molecules, the theory shows that the unfolded state has a much higher density than a chain in a theta solvent and that the density increases with ionic strength. In addition, from the free energy of proton binding to the protein, we also calculate the electrostatic contributions to protein stability, a major contribution deriving from changes in ionization. We consider the example of the stability of myoglobin as a function of pH, ionic strength, and ionic groups buried in the native protein structure. We show that although maximum stability of most proteins should occur at their isoelectric point, the burial of nontitratable groups should lead to maximum stabilities at pH values other than the isoelectric point.     

How orientational order governs collectivity of folded proteins

The past decade has witnessed the development and success of coarse‐grained network models of proteins for predicting many equilibrium properties related to collective modes of motion. Curiously, the results are usually robust toward the different cutoff distances used for constructing the residue networks from the knowledge of the experimental coordinates. In this study, we present a systematical study of network construction and their effect on the predicted properties. Probing bond orientational order around each residue, we propose a natural partitioning of the interactions into an essential and a residual set. In this picture, the robustness originates from the way with which new contacts are added, so that an unusual local orientational order builds up. These residual interactions have a vanishingly small effect on the force vectors on each residue. The stability of the overall force balance then translates into the Hessian as small shifts in the slow modes of motion and an invariance of the corresponding eigenvectors. We introduce a rescaled version of the Hessian matrix and point out a link between the matrix Frobenius norm based on spectral stability arguments and orientational local order. A recipe for the optimal choice of partitioning the interactions into essential and residual components is prescribed. Implications for the study of biologically relevant properties of proteins are discussed with specific examples. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Import of stably folded proteins into peroxisomes.

By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse the single membrane of this organelle. Because the targeting signal for most peroxisomal matrix proteins is a carboxy-terminal tripeptide sequence (SKL or its variants), these proteins must remain import competent until their translation is complete. We sought to determine whether stably folded proteins were substrates for peroxisomal import. Prefolded proteins stabilized with disulfide bonds and chemical cross-linkers were shown to be substrates for peroxisomal import, as were mature folded and disulfide-bonded IgG molecules containing the peroxisomal targeting signal. In addition, colloidal gold particles conjugated to proteins bearing the peroxisomal targeting signal were translocated into the peroxisomal matrix. These results support the concept that proteins may fold in the mammalian cytosol, before their import into the peroxisome, and that protein unfolding is not a prerequisite for peroxisomal import.     

Macromolecular crowding increases structural content of folded proteins

Here we show that increased amount of secondary structure is acquired in the folded states of two structurally-different proteins (alpha-helical VlsE and alpha/beta flavodoxin) in the presence of macromolecular crowding agents. The structural content of flavodoxin and VlsE is enhanced by 33% and 70%, respectively, in 400 mg/ml Ficoll 70 (pH 7, 20 degrees C) and correlates with higher protein-thermal stability. In the same Ficoll range, there are only small effects on the unfolded-state structures of the proteins. This is the first in vitro assessment of crowding effects on the native-state structures at physiological conditions. Our findings imply that for proteins with low intrinsic stability, the functional structures in vivo may differ from those observed in dilute buffers.     

The nature of folded states of globular proteins.

We suggest, using dynamical simulations of a simple heteropolymer modelling the alpha-carbon sequence in a protein, that generically the folded states of globular proteins correspond to statistically well-defined metastable states. This hypothesis, called the metastability hypothesis, states that there are several free energy minima separated by barriers of various heights such that the folded conformations of a polypeptide chain in each of the minima have similar structural characteristics but have different energies from one another. The calculated structural characteristics, such as bond angle and dihedral angle distribution functions, are assumed to arise from only those configurations belonging to a given minimum. The validity of this hypothesis is illustrated by simulations of a continuum model of a heteropolymer whose low temperature state is a well-defined beta-barrel structure. The simulations were done using a molecular dynamics algorithm (referred to as the "noisy" molecular dynamics method) containing both friction and noise terms. It is shown that for this model there are several distinct metastable minima in which the structural features are similar. Several new methods of analyzing fluctuations in structures belonging to two distinct minima are introduced. The most notable one is a dynamic measure of compactness that can in principle provide the time required for maximal compactness to be achieved. The analysis shows that for a given metastable state in which the protein has a well-defined folded structure the transition to a state of higher compactness occurs very slowly, lending credence to the notion that the system encounters a late barrier in the process of folding to the most compact structure. The examination of the fluctuations in the structures near the unfolding----folding transition temperature indicates that the transition state for the unfolding to folding process occurs closer to the folded state.     

H-bonding mediates polarization of peptide groups in folded proteins

The carbon-nitrogen J-couplings in the hydrogen bonding chains of proteins show that H-bonding mediates peptide-group polarization, which results in the general reduction of peptide-group polarity of folded proteins in solution. The net effect is to make large regions of protein secondary structure, especially beta-sheets, intrinsically more hydrophobic, contributing thereby to overall stability of the tertiary structure.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

Complement yourself: transcomplementation rescues partially folded mutant proteins

Cystic fibrosis (CF) is an autosomal disease associated with malfunction in fluid and electrolyte transport across several mucosal membranes. The most common mutation in CF is an in-frame three-base pair deletion that removes a phenylalanine at position 508 in the first nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) chloride channel. This mutation has been studied extensively and leads to biosynthetic arrest of the protein in the endoplasmic reticulum and severely reduced channel activity. This review discusses a novel method of rescuing ΔF508 with transcomplementation, which occurs when smaller fragments of CFTR containing the wild-type nucleotide binding domain are co-expressed with the F508 deletion mutant. Transcomplementation rescues the processing and channel activity of ΔF508 and reduces its rate of degradation in airway epithelial cells. To apply transcomplementation as a therapy would require that the cDNA encoding the truncated CFTR be delivered to cells. We also discuss a gene therapeutic approach based on delivery of a truncated form of CFTR to airway cells using adeno-associated viral vectors.     

Expanding the proteome: disordered and alternatively folded proteins

Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional (3D) structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question 'why would a particular domain need to be unstructured?' are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but also of partially structured and highly dynamic members of the disorder-order continuum.     

Correctly folded proteins make twice as many hydrophobic contacts

A novel statistical analysis of non-bonded contacts in a set of known protein structures shows that the natural residue types fall into five or six groups distinguishable by nearest neighbor preference. The observed pattern of contact specificities clearly reflects residue hydrophobicity and charge. Its most striking feature is that residues in the hydrophobic group make about twice as many contacts with one another as would be expected on a random basis. A similar increase in hydrophobic contact frequency can be observed at the level of individual proteins. Native proteins make, on average, about twice as many hydrophobic contacts as corresponding misfolded proteins, generated by computer. On the basis of these observations increased hydrophobic contact frequency is proposed as a simple model of the hydrophobic effect.     

High-pressure SAXS study of folded and unfolded ensembles of proteins

A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.     

The role of packing interactions in stabilizing folded proteins.

In order to investigate the role of nonpolar side chains in determining protein stability, we have carried out a molecular dynamics simulation study of the thermodynamics of interconverting isoleucine and valine side chains in the core of ribonuclease T1. The free energy change in the unfolded state, which we take to be fully solvated, was small and agrees qualitatively with experimental studies of alkane solvation. In the two Ile----Val mutations studied, the protein was able to relax around the smaller side chains, while in the case of the two Val----Ile mutations, the ability of the core to accommodate the extra methylene group depended on where the mutation took place. We argue that the experimentally observed decrease in stability for mutating isoleucine into valine results from a loss of favorable packing interactions of the side chain in the folded form of the protein. This supports the view that packing interactions in the folded state are an important contributor to the overall stability of the folded protein and that the core of the native protein is packed efficiently and almost completely.     

Amyloid oligomer conformation in a group of natively folded proteins

Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic beta-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-beta-aggregation activity in terms of both functionality and in contrast to the beta-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-beta-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-beta-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Abeta aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the beta-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the beta-sheet edge in some natively folded amyloid oligomers is designed positively to prevent beta aggregation.