Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

Derivation of a solubility condition for proteins from an analysis of the competition between folding and aggregation

Failure in maintaining protein solubility in vivo impairs protein homeostasis and results in protein misfolding and aggregation, which are often associated with severe neurodegenerative and systemic disorders that include Alzheimer's and Parkinson's diseases and type II diabetes. In this work we formulate a model of the competition between folding and aggregation, and derive a condition on the solubility of proteins in terms of the stability of their folded states, their aggregation propensities and their degradation rates. From our model, the bistability between folding and aggregation emerges as an intrinsic aspect of protein homeostasis. The analysis of the conditions that determine such a bistability provides a rationalization of the recently observed relationship between the cellular abundance and the aggregation propensity of proteins. We then discuss how the solubility condition that we derive can help rationalise the correlation that has been reported between evolutionary rates and expression levels or proteins, as well as in vivo protein solubility and expression level measurements, and recently elucidated trends of proteome evolution.     

Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding

The eukaryotic chaperonin TRiC/CCT mediates folding of an essential subset of newly synthesized proteins, including the tumor suppressor VHL. Here we show that chaperonin binding is specified by two short hydrophobic beta strands in VHL that, upon folding, become buried within the native structure. These TRiC binding determinants are disrupted by tumor-causing point mutations that interfere with chaperonin association and lead to misfolding. Strikingly, while unable to fold correctly in vivo, some of these VHL mutants can reach the native state when refolded in a chaperonin-independent manner. The specificity of TRiC/CCT for extended hydrophobic beta strands may help explain its role in folding aggregation-prone polypeptides. Our findings reveal a class of disease-causing mutations that inactivate protein function by disrupting chaperone-mediated folding in vivo.     

In vivo fragment complementation of a (beta/alpha)(8) barrel protein: generation of variability by recombination

The high representation of the TIM barrel as a scaffold for enzymatic proteins makes it an interesting model for protein engineering. Based on previous reports of folding mechanisms of TIM barrels that suggest an independent folding unit formed by six (beta/alpha) subunits, we interrupted the gene of phosphoribosylanthranilate isomerase (PRAI) from Escherichia coli at three different positions to yield fragments with different combinations of (beta/alpha) subunits. When these constructions were expressed as polycistrons in a TrpF-E. coli strain, complementation of the function only occurred with fragments beta1-alpha4 and beta5-alpha8, demonstrating that (beta/alpha)(4) subunits are stable enough to survive in vivo conditions and to assemble to yield a functional enzyme. The expression of these fragments in a separated plasmid/phagemid system to complement the function gave a slower complementation in the TrpF-E. coli strain; this was overcome by introducing extra secondary elements to the structure that reinforce their interaction.     

Genetic screens and directed evolution for protein solubility

Overexpressed proteins are often insoluble, and can be recalcitrant to conventional solubilization techniques such as refolding. Directed evolution methods, in which protein diversity libraries are screened for soluble variants, offer an alternative route to obtaining soluble proteins. Recently, several new protein solubility screens have been developed that do not require structural or functional information about the target protein. Soluble protein can be detected in vivo and in vitro by fusion reporter tags. Protein misfolding can be measured in vivo using the bacterial response to protein misfolding. Finally, soluble protein can be monitored by immunological detection. Efficient, well-established strategies for generating and recombining genetic diversity, driven by new screening and selection methods, can furnish correctly folded, soluble protein.     

Protein solubility and folding enhancement by interaction with RNA

While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.     

Monitoring protein stability <Emphasis Type="Italic">in vivo</Emphasis>

Reduced protein stability in vivo is a prerequisite to aggregation. While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit.     

A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein

Protein misfolding is now recognized as playing a crucial role in both normal and pathogenic folding reactions. An interesting example of misfolding at the earliest state of a natural folding reaction is provided by the alpha-subunit of tryptophan synthase, a (beta/alpha)(8) TIM barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, I(BP), and a subsequent on-pathway intermediate, I1, was probed by mutational analysis of 20 branched aliphatic side-chains distributed throughout the sequence. The elimination of I(BP) and the substantial destabilization of I1 by replacement of a selective set of the isoleucine, leucine or valine residues (ILV) with alanine in a large ILV cluster external-to-the-barrel and spanning the N and C termini (cluster 2) implies tight-packing at most sites in both intermediates. Differential effects on I(BP) and I1 for replacements in alpha3, beta4 and alpha8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not I(BP) reflects non-native folds at the edges of the crucial (beta/alpha)(1-2)beta(3) core in I(BP). The retention of I(BP) and the smaller and consistent destabilization of both I(BP) and I1 by similar replacements in an internal-to-the-barrel ILV cluster (cluster 1) and a second external-to-the-barrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for I(BP) or for all of I1 in cluster 2, but not in clusters 1 and 3, may reflect the larger size of cluster 2 and/or the enhanced number of isoleucine, leucine and valine self-contacts in and between contiguous elements of secondary structure. Tightly packed ILV-dominated hydrophobic clusters could serve as an important driving force for the earliest events in the folding and misfolding of the TIM barrel and other members of the (beta/alpha)(n) class of proteins.     

Misfolding of membrane proteins in health and disease: the lady or the tiger?

Protein misfolding is increasingly recognized as a factor in many diseases, including cystic fibrosis, Parkinson's, Alzheimer's and atherosclerosis. Many proteins involved in misfolding-based pathologies are membrane-associated, such that the bilayer may play roles in normal and aberrant folding. It can be argued that the in vivo partitioning of eukaryotic membrane proteins between folding and misfolding pathways is under kinetic control. Moreover, the balance between these pathways can be surprisingly delicate.     

A screen to identify cellular modulators of soluble levels of an amyotrophic lateral sclerosis (ALS)-causing mutant SOD1

The molecular pathology of many protein misfolding, toxic gain-of-function diseases, such as amyotrophic lateral sclerosis (ALS), is not well understood. Although protein misfolding and aggregation are common themes in these diseases, efforts to identify cellular factors that regulate this process in an unbiased fashion and on a global scale have been lacking. Using an adapted version of an extant β-gal-based protein solubility assay, an expression screen for cellular modulators of solubility of an ALS-causing mutant SOD1 was carried out in mammalian cells. Following fluorescence-activated cell sorting enrichment of a mouse spinal cord cDNA library for gene products that increased SOD1 solubility, high-throughput screening of the cDNA pools from this enriched fraction was employed to identify pools containing relevant modulators. Positive pools, containing approximately 10 cDNA clones each, were diluted and rescreened iteratively until individual clones that improved SOD1 folding/solubility were identified. Genes with profound effects in the solubility assay were selected for validation by independent biochemical assays. Six of 10 validated genes had a significant effect on SOD1 solubility and folding in a SOD1 promoter-driven β-gal assay, indicating that global screening of cellular targets using such protein solubility/folding assay is viable and can be adapted for other misfolding diseases.     

The dual role of a loop with low loop contact distance in folding and domain swapping

Alpha helices, beta strands, and loops are the basic building blocks of protein structure. The folding kinetics of alpha helices and beta strands have been investigated extensively. However, little is known about the formation of loops. Experimental studies show that for some proteins, the formation of a single loop is the rate-determining step for folding, whereas for others, a loop (or turn) can misfold to serve as the hinge loop region for domain-swapped species. Computer simulations of an all-atom model of fragment B of Staphylococcal protein A found that the formation of a single loop initiates the dominant folding pathway. On the other hand, the stability analysis of intermediates suggests that the same loop is a likely candidate to serve as a hinge loop for domain swapping. To interpret the simulation result, we developed a simple structural parameter: the loop contact distance (LCD), or the sequence distance of contacting residues between a loop and the rest of the protein. The parameter is applied to a number of other proteins, including SH3 domains and prion protein. The results suggest that a locally interacting loop (low LCD) can either promote folding or serve as the hinge region for domain swapping. Thus, there is an intimate connection between folding and domain swapping, a possible cause of misfolding and aggregation.     

Homogeneous sandwich immunoassay based on the enzymatic complementation induced by single-chain Fv fragments

We describe a novel homogeneous sandwich immunoassay based on beta-galactosidase (beta-gal) complementation (the crab-claw sandwich enzymatic complementation immunoassay, CS-ECIA). We chose a high-molecular-weight antigen human serum albumin (HSA) as a model and constructed two chimeric proteins, in which a pair of single-chain Fvs (scFvs) recognizing distant epitopes of HSA was fused to either an N-terminal deletion mutant of beta-gal (deltaalpha) or a C-terminal deletion mutant of beta-gal (deltaomega). Upon simple mixing of the reagents with the sample, the two chimeric proteins became associated through binding separate epitopes on HSA that allowed reassociation of the two mutant enzymes. The resulting enzymatic complementation was measured as an increase in beta-gal activity using a luminescent substrate. With this CS-ECIA, a HSA concentration of 10-1000 pg/mL could be determined. In addition, the assay was easy to operate and required less time, handling, and sample volume than conventional sandwich enzyme-linked immunoassays. The assay will have general utility by substituting scFvs with other pairs of scFvs recognizing any polyvalent antigens.     

Entropy capacity determines protein folding

Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Here, based on the known experimental data and using theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue-that is, an entropy capacity-for fast protein folding. Statistical analysis of conformational entropy and number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts (class alpha/beta has the largest number of contacts) and average conformational entropy per residue (class all-alpha has the largest number of rotatable angles phi, psi, and chi per residue). These class-specific features determine the folding rates: alpha proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones. Our result is in agreement with the experimental folding rates for 60 proteins. This suggests that structural and sequence properties are important determinants of protein folding rates.     

Productive interaction of chaperones with substrate protein domains allows correct folding of the downstream GFP domain

Green fluorescent protein (GFP) has been used to report protein folding by correlating solubility with fluorescence. In a GFP fusion protein, an upstream aggregation-prone domain can disrupt de novo folding of the GFP domain in Escherichia coli, resulting in a loss of fluorescence. Previously, we showed that prevention of misfolding of the upstream aggregation-prone domain by a coupled folding and binding interaction during protein synthesis restored both GFP fluorescence and solubility. Since molecular chaperones often fold nascent polypeptides through a bind-and-release interaction, the question remains whether the chaperone interaction with the upstream aggregation-prone domain enhances GFP fluorescence. Here, we demonstrate that a significant increase in GFP fluorescence occurred only when appropriate chaperones that recognized the aggregation-prone protein and helped its folding were co-expressed. A possible correlation between GFP fluorescence and the productive folding by chaperones is proposed. This study may provide a general strategy for identifying chaperones specific for difficult-to-fold proteins.     

Mining mammalian genomes for folding competent proteins using Tat‐dependent genetic selection in Escherichia coli

Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin‐arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N‐terminal Tat export signal and a C‐terminal selectable marker, namely β‐lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein.     

Optimal relationship between average conformational entropy and average energy of interactions between residues for fast protein folding

Based on the known experimental data and using the theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue, that is an entropy capacity, for fast protein folding. Statistical analysis of conformational entropy and the number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, +/-/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts and average conformational entropy per residue. These class-specific features determine the folding rates: a proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones.     

Observing folding pathways and kinetics of a single sodium-proton antiporter from Escherichia coli

Mechanisms of folding and misfolding of membrane proteins are of interest in cell biology. Recently, we have established single-molecule force spectroscopy to observe directly the stepwise folding of the Na+/H+ antiporter NhaA from Escherichia coli in vitro. Here, we improved this approach significantly to track the folding intermediates of a single NhaA polypeptide forming structural segments such as the Na+-binding site, transmembrane alpha-helices, and helical pairs. The folding rates of structural segments ranged from 0.31 s(-1) to 47 s(-1), providing detailed insight into a distinct folding hierarchy of an unfolded polypeptide into the native membrane protein structure. In some cases, however, the folding chain formed stable and kinetically trapped non-native structures, which could be assigned to misfolding events of the antiporter.     

Signatures of co-translational folding

Global and co-translational protein folding may both occur in vivo, and understanding the relationship between these folding mechanisms is pivotal to our understanding of protein-structure formation. Within this study, over 1.5 million hydrophobic-polar sequences were classified based on their ability to attain a unique, but not necessarily minimal energy conformation through co-translational folding. The sequence and structure properties of the sets were then compared to elucidate signatures of co-translational folding. The strongest signature of co-translational folding is a reduced number of possible favorable contacts in the amino terminus. There is no evidence of fewer contacts, more local contacts, or less-compact structures. Co-translational folding produces a more compact amino- than carboxy-terminal region and an amino-terminal-biased set of core residues. In real proteins these signatures are also observed and found most strongly in proteins of the alpha/beta structural class of proteins (SCOP) where 71 % have an amino-terminal set of core residues. The prominence of co-translational features in experimentally determined protein structures suggests that the importance of co-translational folding is currently underestimated.     

New molecular reporters for rapid protein folding assays

The GFP folding reporter assay uses a C-terminal GFP fusion to report on the folding success of upstream fused polypeptides. The GFP folding assay is widely-used for screening protein variants with improved folding and solubility, but truncation artifacts may arise during evolution, i.e. from de novo internal ribosome entry sites. One way to reduce such artifacts would be to insert target genes within the scaffolding of GFP circular permuted variants. Circular permutants of fluorescent proteins often misfold and are non-fluorescent, and do not readily tolerate fused polypeptides within the fluorescent protein scaffolding. To overcome these limitations, and to increase the dynamic range for reporting on protein misfolding, we have created eight GFP insertion reporters with different sensitivities to protein misfolding using chimeras of two previously described GFP variants, the GFP folding reporter and the robustly-folding "superfolder" GFP. We applied this technology to engineer soluble variants of Rv0113, a protein from Mycobacterium tuberculosis initially expressed as inclusion bodies in Escherichia coli. Using GFP insertion reporters with increasing stringency for each cycle of mutagenesis and selection led to a variant that produced large amounts of soluble protein at 37 degrees C in Escherichia coli. The new reporter constructs discriminate against truncation artifacts previously isolated during directed evolution of Rv0113 using the original C-terminal GFP folding reporter. Using GFP insertion reporters with variable stringency should prove useful for engineering protein variants with improved folding and solubility, while reducing the number of artifacts arising from internal cryptic ribosome initiation sites.