Folding Efficiency Research Articles

Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide‐Rich Peptides

AbstractDisulfide‐rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low‐yielding folding process. We found that a single cystine‐to‐diaminodiacid replacement could significantly increase the folding efficiency of disulfide‐rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ‐conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI‐II. NMR and X‐ray crystallography studies confirmed that these derivatives of disulfide‐rich peptide retained the correct three‐dimensional conformations. Moreover, the cystine‐to‐diaminodiacid replacement enabled structural tuning, thereby leading to an EETI‐II derivative with higher bioactivity than the native peptide.

Peptide Folding in Translocon-Like Pores.

The cellular translocon, present in all three domains of life, is one of the most versatile and important biological nanopores. This complex molecular apparatus is directly responsible for the secretion of globular proteins across membranes as well as the insertion of integral membrane proteins into lipid bilayers. Recently determined structures of the archaean SecY translocon reveal an hour-glass-shaped pore, which accommodates the nascent peptide chain during translocation. While these structures provide important insights into ribosome binding to the translocon, threading of the nascent chain into the channel, and lateral gate opening for releasing the folded helical peptide into the membrane bilayer, the exact folding pathway of the peptide inside the protein-conducting channel during translocation and prior to the lateral release into the bilayer remains elusive. In the present study, we use molecular dynamics simulations to investigate atomic resolution peptide folding in hour-glass-shaped pore models that are based on the SecY translocon channel structure. The theoretical setup allows systematic variation of key determinants of folding, in particular the degree of confinement of the peptide and the hydration level of the pore. A 27-residue hydrophobic peptide was studied that is preferentially inserted into membranes by the translocon. Our results show that both pore diameter as well as channel hydration are important determinants for folding efficiency and helical stability of the peptide, therefore providing important insights into translocon gating and lateral peptide partitioning.

Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.

Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors.

Molecular pathways: the PERKs and pitfalls of targeting the unfolded protein response in cancer.

The endoplasmic reticulum (ER) is a highly specialized organelle that provides an oxidizing, profolding environment for protein synthesis and maturation. The ER also hosts a dynamic signaling network that can sense and respond to physiologic changes that affect its environment, thereby influencing overall cell fate. Limitation of nutrients and oxygen have a direct effect on the efficiency of protein folding in the ER, and are classic inducers of the ER resident signaling pathway, the unfolded protein response (UPR). Not only does the UPR regulate ER homeostasis in normal cells experiencing such stress, but strong evidence also suggests that tumor cells can co-opt the cytoprotective aspects of this response to survive the hypoxic, nutrient-restricted conditions of the tumor microenvironment.

CCT5: A Model Protein Folding Machine

The chaperonin TRiC is a 16-subunit protein complex, composed of two rings, with 8 individual subunits each (CCT1-8), that functions as a protein folding machine in eukaryotic cells. TRiC is required for the folding of approximately 10% of the human proteome, including a number of crucial cellular proteins, most notably actin and tubulin. While TRiC-mediated folding is critical for many cellular processes, its underlying mechanism is poorly understood. Mechanistic and structural elucidation of TRiC folding activity is hampered in large part by the sheer size and subunit heterogeneity of the chaperonin, and therefore most studies have focused on the functionally related but structurally less-complex chaperonins, including the thermosome and Mm-Cpn, as surrogates for TRiC architecture and function. Recently, Sergeeva et al demonstrated that human CCT4 and CCT5 subunits expressed individually in E. coli form homo-oligimeric chaperonins capable of folding native substrates. Here, we have used purified CCT5 homo-oligomer to investigate the in vitro chaperonin-dependent folding of human gD-crystallin and beta-actin. Modifications to the CCT5 primary sequence, including changes to the N and C termini, resulted in changes in folding efficiency, substrate recognition and suppression of aggregation. Structural, biochemical and computational studies on these modifications will provide insight into the mechanism of the TRiC folding machine.

Chemical Regulation of Disulfide Coupled Folding of Disulfide Rich Peptide, Hepcidin, and its Precursor Protein

Multi disulfide-containing-proteins are folded into the native conformations via the assistance of redox molecules, such as glutathione and cysteine in vivo. Because of this, the glutathione redox system is also generally used in in vitro folding studies [1]. However, in vitro disulfide-coupled peptide and protein folding are often problematic, since the folding intermediates are sometimes prone to aggregation. In this study, to overcome this issue and chemically regulate disulfide-coupled peptide and protein folding, a series of chemical reagents was examined in the refolding of hepcidin and prohepcidin, as a model peptide and protein, and their folding recoveries were estimated.Hepcidin consists of 25 amino acid residues and four intra-molecular disulfide bonds, which are absolutely required for its biological activity [2], not only for iron homeostasis, but also for anti-microbial activity. To investigate structure-function relationships, hepcidin was chemically synthesized. However, the yield of synthesized hepcidin was quite low under the typical folding conditions. The major problem in the disulfide-coupled folding of hepcidin is that it undergoes aggregation during its folding reaction [3]. To solve this problem, several types of redox reagents and solvents were examined to improve the folding efficiency of hepcidin [3]. However, all of the reagents resulted in quite low yields for the disulfide-coupled folding of hepcidin. Therefore, to regulate the folding reaction of hepcidin and its precursor protein, we estimated the folding conditions, such as pH values, salt concentrations, and a variety of redox reagents. The results will be discussed in this paper.[1] Chakravarthi, S.; Jessop, C.E.; Bulleid, N.J. EMBO reports 2006, 7, 271-275.[2] Hocquellet, A.; le Senechal, C.; Garbay, B. Peptides 2012, 36, 303-307.[3] Zhang, J.; Diamond, S.; Arvedson, T.; Sasu, BJ.; Miranda, LP. Biopolymers 2010, 94, 257-264.

The Role of a Destabilized Membrane for OMP Insertion.

Here we describe the procedures used in our laboratory for the in vitro investigation of the apparent folding kinetics as well as the folding efficiencies of outer membrane proteins (OMPs). Because microbial OMPs display a change in their gel migration upon folding, the usage of traditional gel electrophoresis is a standard method of folding analysis. Additional aspects of the method we detail herein include the preparation and storage of OMP stocks, the setup procedures for a folding reaction, and the analysis of fraction folded from scanned gel images.

Structural and functional study of the GlnB22-insulin mutant responsible for maturity-onset diabetes of the young.

The insulin gene mutation c.137G>A (R46Q), which changes an arginine at the B22 position of the mature hormone to glutamine, causes the monogenic diabetes variant maturity-onset diabetes of the young (MODY). In MODY patients, this mutation is heterozygous, and both mutant and wild-type (WT) human insulin are produced simultaneously. However, the patients often depend on administration of exogenous insulin. In this study, we chemically synthesized the MODY mutant [GlnB22]-insulin and characterized its biological and structural properties. The chemical synthesis of this insulin analogue revealed that its folding ability is severely impaired. In vitro and in vivo tests showed that its binding affinity and biological activity are reduced (both approximately 20% that of human insulin). Comparison of the solution structure of [GlnB22]-insulin with the solution structure of native human insulin revealed that the most significant structural effect of the mutation is distortion of the B20-B23 β-turn, leading to liberation of the B chain C-terminus from the protein core. The distortion of the B20-B23 β-turn is caused by the extended conformational freedom of the GlnB22 side chain, which is no longer anchored in a hydrogen bonding network like the native ArgB22. The partially disordered [GlnB22]-insulin structure appears to be one reason for the reduced binding potency of this mutant and may also be responsible for its low folding efficiency in vivo. The altered orientation and flexibility of the B20-B23 β-turn may interfere with the formation of disulfide bonds in proinsulin bearing the R46Q (GlnB22) mutation. This may also have a negative effect on the WT proinsulin simultaneously biosynthesized in β-cells and therefore play a major role in the development of MODY in patients producing [GlnB22]-insulin.

Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration.

An R345W mutation in the N-glycoprotein, fibulin-3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N-Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N-glycosylation alters the protein homeostasis or proteostasis of wild-type (WT) and R345W F3 in ARPE-19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N-glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N-glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N-glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variant's unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N-glycosylation to acquire a stable, native-like structure.

The influenza virus neuraminidase protein transmembrane and head domains have coevolved.

Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept, as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly more polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical "old" TMD (1933) with a "recent" (2009), more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37°C compared to at 33°C, at which NA folds more efficiently. Passaging the chimera viruses at 37°C restored the NA folding efficiency, viral budding, and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggest the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain. The neuraminidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor-destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an "old" N1 head domain from 1933 is incompatible with a "recent" (2009), more polar N1 TMD sequence and that, during viral replication, the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have coevolved.

The lower limits for protein stability and foldability in primary hyperoxaluria type I

Mutational effects on protein stability and foldability are important to understand conformational diseases and protein evolution. In this work, we perform a comprehensive investigation on the energetic basis underlying mutational effects on the stability of human alanine:glyoxylate aminotransferase (AGT). We study twenty two variants whose kinetic stabilities span over eleven orders of magnitude and are classified into two groups: i) ten naturally-occurring variants, including the most common mutations causing primary hyperoxaluria type I (PH1); and ii) twelve consensus variants obtained by sequence-alignment statistics. We show that AGT dimer stability determines denaturation rates, and mutations modulate stability by changes in the effective thermodynamic stability, the aggregation propensity of partially/globally unfolded states and subtle energetic changes in the rate-limiting denaturation step. In combination with our previous expression analyses in eukaryotic cells, we propose the existence of two lower limits for AGT stability, one linked to optimal folding efficiency (close to the major allele stability) and the other setting a minimal efficiency compatible with glyoxylate detoxification in vivo (close to the minor allele stability). These lower limits could explain the high prevalence of misfolding as a disease mechanism in PH1 and support the use of pharmacological ligands aimed to increase AGT stability as therapies for this disease.

Predicting aggregation-prone sequences in proteins.

Owing to its association with a diverse range of human diseases, the determinants of protein aggregation are studied intensively. It is generally accepted that the effective aggregation tendency of a protein depends on many factors such as folding efficiency towards the native state, thermodynamic stability of that conformation, intrinsic aggregation propensity of the polypeptide sequence and its ability to be recognized by the protein quality control system. The intrinsic aggregation propensity of a polypeptide sequence is related to the presence of short APRs (aggregation-prone regions) that self-associate to form intermolecular β-structured assemblies. These are typically short sequence segments (5-15 amino acids) that display high hydrophobicity, low net charge and a high tendency to form β-structures. As the presence of such APRs is a prerequisite for aggregation, a plethora of methods have been developed to identify APRs in amino acid sequences. In the present chapter, the methodological basis of these approaches is discussed, as well as some practical applications.

Salicylic Acid Signaling Controls the Maturation and Localization of the Arabidopsis Defense Protein ACCELERATED CELL DEATH6

ACCELERATED CELL DEATH6 (ACD6) is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid (SA). This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum (ER)- and plasma membrane (PM)-localized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses.

Effects of knot type in the folding of topologically complex lattice proteins.

The folding properties of a protein whose native structure contains a 52 knot are investigated by means of extensive Monte Carlo simulations of a simple lattice model and compared with those of a 31 knot. A 52 knot embedded in the native structure enhances the kinetic stability of the carrier lattice protein in a way that is clearly more pronounced than in the case of the 31 knot. However, this happens at the expense of a severe loss in folding efficiency, an observation that is consistent with the relative abundance of 31 and 52 knots in the Protein Data Bank. The folding mechanism of the 52 knot shares with that of the 31 knot the occurrence of a threading movement of the chain terminus that lays closer to the knotted core. However, co-concomitant knotting and folding in the 52 knot occurs with negligible probability, in sharp contrast to what is observed for the 31 knot. The study of several single point mutations highlights the importance in the folding of knotted proteins of the so-called structural mutations (i.e., energetic perturbations of native interactions between residues that are critical for knotting but not for folding). On the other hand, the present study predicts that mutations that perturb the folding transition state may significantly enhance the kinetic stability of knotted proteins provided they involve residues located within the knotted core.

BiP clustering facilitates protein folding in the endoplasmic reticulum.

The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as ‘clusters’). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.

Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin.

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin–insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1–B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the “classical” T-state and that a substantial flexibility of the B1–B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin–IR interaction.

A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies.

BackgroundInclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology.ResultsIn this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn’t affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100–200 nm by reducing the expression time of the protein in vivo.ConclusionsTo our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.

Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications

Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolution, resulting in superior probes for use in live-cell fluorescence microscopy. In this protocol, we provide methods for engineering a monomeric FP, for enhancing its brightness by directed evolution, and for thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features, such as a red-shifted emission spectrum or enhanced photostability. Although the length of the procedure is dependent on the degree of optimization desired, the basic steps can be accomplished in 4-6 weeks.

Chemical Synthesis of Insulin Analogs through a Novel Precursor

Insulin remains a challenging synthetic target due in large part to its two-chain, disulfide-constrained structure. Biomimetic single chain precursors inspired by proinsulin that utilize short peptides to join the A and B chains can dramatically enhance folding efficiency. Systematic chemical analysis of insulin precursors using an optimized synthetic protocol identified a 49 amino acid peptide named DesDi, which folds with high efficiency by virtue of an optimized structure and could be proteolytically converted to bioactive two-chain insulin. In subsequent applications, we observed that the folding of the DesDi precursor was highly tolerant to amino acid substitution at various insulin residues. The versatility of DesDi as a synthetic insulin precursor was demonstrated through the preparation of several alanine mutants (A10, A16, A18, B12, B15), as well as ValA16, an analog that was unattainable in prior reports. In vitro bioanalysis highlighted the importance of the native, hydrophobic residues at A16 and B15 as part of the core structure of the hormone and revealed the significance of the A18 residue to receptor selectivity. We propose that the DesDi precursor is a versatile synthetic intermediate for the preparation of diverse insulin analogs. It should enable a more comprehensive analysis of function to insulin structure than might not be otherwise possible through conventional approaches.

Enhanced Chaperone Clustering Facilitates Protein Folding in the Endoplasmic Reticulum of Yeast

The chaperone BiP plays several roles in the endoplasmic reticulum (ER): translocation, protein folding, ER-associated degradation, and a modulating function in Ire1p-regulated ER stress. Experimental evidence has suggested the existence of BiP heterogeneity in the ER. A cooperative mechanism known as entropic pulling has been proposed to explain how the molecular interaction of multiple BiP molecules on unfolded proteins occurs. We have developed a model to explore the potential advantages of the binding of multiple BiP molecules in the facilitation of protein folding in the ER to explain the heterogeneity, and take advantage of entropic pulling.Simulation scenarios were enacted to gauge the effectiveness of multiple chaperone binding in protein folding. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model had a higher efficiency than multiple binding models, in the absence of cooperativity. Through entropic pulling, however, multiple chaperones do work in concert to facilitate the resolubilization and ultimate yield of folded proteins. Under a cooperative scenario, multiple binding models used fewer chaperones and enjoyed a higher folding efficiency than the single binding site model.In conclusion, our in-silico experiments reveal that clusters of BiP molecules bound to unfolded proteins could enhance folding efficiency through cooperative action via entropic pulling.