Rate Of Refolding Research Articles

Quantitative analytics for protein refolding states

Development and optimization of inclusion body (IB) refolding processes is often based on empirical strategies. Thus, generation of in-depth process understanding is crucial to shift towards more knowledge-driven approaches. Within this contribution the goal was to establish a systematic approach for the reliable quantification of process dynamics in protein refolding processes. Established offline- analytical tools like HPLC-based methods or photometric assays were analyzed regarding their specifications and consequent suitability to quantify protein states. An in-silico study was conducted to define requirements for state quantification by analyzing the influences of error, discrete sampling intervals and the pace of the reaction on signal quality. Refolding and aggregation reaction rates were calculated by finite difference approximation with errors propagated from the corresponding folding state. Application of the defined principles on two real Lactate dehydrogenase fed-batch refolding processes resulted in two individual sampling strategies. At reaction rates of up to 0.58 g h−1 description of the ongoing dynamics could be done accurately with high sampling intervals during the first few hours of processing as shown by comparison to a noise free simulation. The proposed method has the potential to be transferred to other proteins and to facilitate the development of model-based monitoring & control strategies.

Immunoglobulin domain unfolding and refolding in passively stretched myofibrils.

In isolated myofibrils, passive force is attributed solely to the protein titin. Mechanically, titin is a non-linear biological spring, with two primary mechanical regions: the PEVK region, and the immunoglobulin (IG) domain region. The PEVK region is approximately elastic, and the IG domain region contains domains that unfold and refold depending on force. Under passive tension, skeletal muscle myofibrils exhibit stress-relaxation, particularly at longer lengths (>3.6 µm/sarcomere) where IG domains begin to unfold. During muscle shortening, IG domains refold, and the PEVK region shortens. We investigated the rate of IG domain unfolding and refolding in myofibrils. Paired tests on myofibrils isolated from the psoas muscle of rabbits (n=7). Passive force was measured using force cantilevers for two test protocols: a 2 s-4 s-2 s stretch, hold, release and a 2 s-0 s-2 s stretch, no-hold, release, both with a target length of 4.5μm/sarcomere. Stretches were performed using a piezoelectric motor. Both test protocols were compared to simulated values using the worm-like chain model. Differences between the two test protocols were apparent specifically in the rate of IG domain refolding. Passive force was present experimentally during the entirety of the two second shortening, with the myofibrils remaining taught over the entire duration of both tests. This behavior was not apparent in simulated tests, where myofibrils were predicted to become slack (no passive force) after 0.79 s and 0.9 s during the 2-s shortening period for the 4-s hold protocol and the no-hold protocol, respectively. This suggests IG domain refolding in myofibrils happens considerably quicker in situ than anticipated from isolated titin testing. Our data show that IG domain refolding at this higher rate likely contributes to substantial positive mechanical work during myofibril shortening for both protocols.

ATP utilization by a DEAD-box protein during refolding of a misfolded group I intron ribozyme

DEAD-box helicase proteins perform ATP-dependent rearrangements of structured RNAs throughout RNA biology. Short RNA helices are unwound in a single ATPase cycle, but the ATP requirement for more complex RNA structural rearrangements is unknown. Here we measure the amount of ATP used for native refolding of a misfolded group I intron ribozyme by CYT-19, a Neurospora crassa DEAD-box protein that functions as a general chaperone for mitochondrial group I introns. By comparing the rates of ATP hydrolysis and ribozyme refolding, we find that several hundred ATP molecules are hydrolyzed during refolding of each ribozyme molecule. After subtracting nonproductive ATP hydrolysis that occurs in the absence of ribozyme refolding, we find that approximately 100 ATPs are hydrolyzed per refolded RNA as a consequence of interactions specific to the misfolded ribozyme. This value is insensitive to changes in ATP and CYT-19 concentration and decreases with decreasing ribozyme stability. Because of earlier findings that ∼90% of global ribozyme unfolding cycles lead back to the kinetically preferred misfolded conformation and are not observed, we estimate that each global unfolding cycle consumes ∼10 ATPs. Our results indicate that CYT-19 functions as a general RNA chaperone by using a stochastic, energy-intensive mechanism to promote RNA unfolding and refolding, suggesting an evolutionary convergence with protein chaperones.

Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP.

Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the process is generally very fast, and we need a suitable read out. In this protocol, we have solved this issue by using a protein that is non-fluorescent in its unfolded state but fluoresces in its native state after folding. The kinetics of refolding can be monitored by following the increase in fluorescence in real time. Previously, this was generally achieved by either monitoring a protein's enzymatic activity or measuring the tryptophan fluorescence, where the signal output depends on well-described enzymatic activity or the frequency of tryptophan residues present in the proteins, respectively. Here, we describe a simple and real-time assay to monitor the refolding of sGFP, a recently described slow-folding mutant of yeGFP (yeast enhanced GFP). We unfold this protein using chemical denaturant and refold in a suitable buffer, monitoring the increase in fluorescence over time. GFP is fluorescent only when correctly folded; thus, using this technique, we can measure the true rate of protein refolding by following the increase in fluorescence over time. Therefore, sGFP can be used as an ideal model to study the in vitro protein folding process. Accordingly, the effects of different conditions and molecules on the protein folding pathway can be efficiently studied using sGFP as a model protein. Graphical abstract: Schematic of the steps involved in the sGFP refolding pathway. Native sGFP is unfolded by chemical denaturation using 6 M GuHCl at 25°C for 1 hour and then refolded in refolding buffer by 100-fold dilution.

HspA1A, a 70‐kDa heat‐shock protein, contains several distinct lipid‐binding sites

HspA1A, a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70), is a molecular chaperone that plays critical roles in protein homeostasis and cell survival. In addition to its functions in protein homeostasis, HspA1A also functions at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although the interactions between HspA1A and lipids have important biological consequences, their mechanistic details remain elusive and unexplored because the amino acids responsible for the lipid binding remain largely uncharacterized and the relationship between the chaperone and lipid‐binding functions remains unknown. To clarify the mechanism of the HspA1A‐lipid interactions, we first characterized the properties of a mutation from Tryptophan to Phenylalanine (W90F), known to affect binding of HspA1A to BMP and a Lysine to Alanine (K71A) mutation, which results in complete loss of the chaperone function. Experiments using the lipid vesicle sedimentation (LVS) method and Surface Plasmon Resonance revealed that although the W90F‐HspA1A binding to BMP was different from the WT, this mutation did not affect the binding to PS. Furthermore, our results revealed that the K71A mutation did not significantly change the binding of HspA1A to lipids under any of the conditions tested. These results allowed us to make two predictions: first, the lipid‐binding sites for PS and BMP are lipid‐specific, and second, the chaperone and lipid‐binding functions are distinct. To support the first prediction, we used a combination of structural superimpositions, sequence alignments, and literature observations and predicted three putative lipid‐binding regions on the HspA1A molecule. We tested these predictions using the LVS assay for 20 single or double mutations spanning two of these regions. Our results revealed that only two of these mutations affected the binding to PS, while a third one altered the binding to BMP. To test the second prediction, we performed a series of experiments to determine the effect of lipid binding to the secondary structure and chaperone functions of HspA1A. We used Circular dichroism spectrometry, measured the release of inorganic phosphate, and determined the rate of refolding of chemically denatured beta‐galactosidase in the presence or absence of particular lipid. These experiments revealed that lipid binding did not alter the secondary structure of the protein and affected neither the rate of ATP hydrolysis nor the rate of protein refolding. Together our findings provide further evidence that HspA1A binds to PS and BMP using different amino acid sites and support the notion that the chaperone and lipid‐binding functions of HspA1A do not overlap. These findings provide the basis for future experiments to test the effects of these mutations on the membrane‐localized functions of HspA1A in cancer and stressed cells.Support or Funding InformationThis project was supported by funds from NIH

Extraction and Refolding Determinants of Chaperone-Driven Aggregated Protein Reactivation

Reactivation of protein aggregates plays a fundamental role in numerous situations, including essential cellular processes, hematological and neurological disorders, and biotechnological applications. The molecular details of the chaperone systems involved are known to a great extent but how the overall reactivation process is achieved has remained unclear. Here, we quantified reactivation over time through a predictive mechanistic model and identified the key parameters that control the overall dynamics. We performed new targeted experiments and analyzed classical data, covering multiple types of non-ordered aggregates, chaperone combinations, and experimental conditions. We found that, irrespective of the behavior observed, the balance of surface disaggregation and refolding in solution universally determines the reactivation dynamics, which is broadly described by two characteristic times. This characterization makes it possible to use activity measurements to accurately infer the underlying loss of aggregated protein and to quantify, for the first time, the refolding rates of the soluble intermediates.

Kinetics of the thermal inactivation and the refolding of bacterial luciferases in Bacillus subtilis and in Escherichia coli differ

Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases of differing thermolability. The kinetics of the thermal inactivation and the refolding of luciferases from Photorhabdus luminescens and Photobacterium leiognathi were compared in Gram negative and Gram positive bacteria. In B. subtilis cells, these luciferases are substantially more thermostable than in Escherichia coli. Thermal inactivation of the thermostable luciferase P. luminescens in B. subtilis at 48.5°С behaves as a first-order reaction. In E.coli, the first order rate constant (Kt) of the thermal inactivation of luciferase in E. coli exceeds that observed in B. subtilis cells 2.9 times. Incubation time dependence curves for the thermal inactivation of the thermolabile luciferase of P. leiognathi luciferase in the cells of E. coli and B. subtilis may be described by first and third order kinetics, respectively. Here we shown that the levels and the rates of refolding of thermally inactivated luciferases in B. subtilis cells are substantially lower that that observed in E. coli. In dnaK-negative strains of B. subtilis, both the rates of thermal inactivation and the efficiency of refolding are similar to that observed in wild-type strains. These experiments point that the role that DnaKJE plays in thermostability of luciferases may be limited to bacterial species resembling E. coli.

Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex

In platelets, the glycoprotein (GP) Ib-IX receptor complex senses blood shear flow and transmits the mechanical signals into platelets. Recently, we have discovered a juxtamembrane mechanosensory domain (MSD) within the GPIbα subunit of GPIb-IX. Mechanical unfolding of the MSD activates GPIb-IX signaling into platelets, leading to their activation and clearance. Using optical tweezer-based single-molecule force measurement, we herein report a systematic biomechanical characterization of the MSD in its native, full-length receptor complex and a recombinant, unglycosylated MSD in isolation. The native MSD unfolds at a resting rate of 9 × 10−3 s−1. Upon exposure to pulling forces, MSD unfolding accelerates exponentially over a force scale of 2.0 pN. Importantly, the unfolded MSD can refold with or without applied forces. The unstressed refolding rate of MSD is ∼17 s−1 and slows exponentially over a force scale of 3.7 pN. Our measurements confirm that the MSD is relatively unstable, with a folding free energy of 7.5 kBT. Because MSD refolding may turn off GPIb-IX’s mechanosensory signals, our results provide a mechanism for the requirement of a continuous pulling force of >15 pN to fully activate GPIb-IX.

The cohesin module is a major determinant of cellulosome mechanical stability

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.

Structural Insights into the Regulation Mechanism of HSP90 by Co-chaperone AHA1

Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of “client” proteins, especially those involved in signaling and regulatory processes. This Hsp90 chaperone machinery is also essential for maintaining cellular protein homeostasis and for dealing with proteotoxic stress. The client maturation cycle is regulated by a large number of co-chaperones that sequentially interact with Hsp90 throughout its ATPase cycle. One of the most important, but poorly understood co-chaperones in eukaryotes is Activator of Hsp90 ATPase homolog 1 (Aha1). As a potent stimulator of the Hsp90 ATPase activity, Aha1 is thought to accelerate client protein remodeling. A knockout of Aha1 in yeast severely compromises the maturation of different Hsp90 client proteins. However, Aha1 overexpression inhibits the rate of Hsp90-dependent refolding of denatured luciferase, as well as cystic fibrosis associated protein CFTR. How Aha1, as an Hsp90 activator, facilitates ATP hydrolysis while inhibiting client maturation remains enigmatic. To better understand this question, we have determined the structure of the full-length 200-kDa Hsp90/Aha1 complex at 3.8 Å resolution using cryo-electron microscopy. When bound to Aha1, the Hsp90 was captured in the closed conformation, similar to the X-ray crystal structure of Hsp90 bound with inhibitory co-chaperone p23. However, the binding interface is completely different from that used by p23 as well as that observed by crystallography of a fragment of Aha bound to a fragment of Hsp90. In our structure, the C-terminal domain of Aha1 stabilizes the Hsp90 closed dimer by bridging the C-terminal domain of one protomer and the middle domain/N-terminal domain of the other protomer. This structure begins to explain the Aha1 inhibitory effect on Hsp90 and also provides a basis for designing co-chaperone specific inhibitors to regulate Hsp90 related diseases.

Evaluating the role of puckering and fluorine atom in stability and folding of fluoroproline containing proteins

In the past decade, numerous studies have been reported that the residue specific incorporation of fluorine containing analogs into protein can enhance the stability of protein. On the other hand, the incorporation of fluoroproline can enhance both stability and refolding rate of recombinant proteins. The objective of this study was to determine the reason behind the enhanced stability and refolding rate of protein by comparing GFP variants containing fluoroproline or hydroxyproline. The fluorine atom of 4-fluoroproline played a significant role in enhancing stability, and Cγ-endo puckering property of (4S)-4-fluoroproline and (4S)-4-hydroxyproline plays a key role in enhancing protein refolding rate.

A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics.

Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths.

Protein refolding from bacterial inclusion bodies is a crucial step for the production of recombinant proteins, but the refolding step often results in significantly lower yields due to aggregation. To prevent aggregation, chemical additives are often used. However, the ability of additives to effectively increase refolding yields are protein dependent, and therefore, it is important to understand the manner in which the substructures of additives confer suitable properties on protein refolding. We focused attention on nonionic detergents, the polyethylene glycol monooleyl ether (PGME) series, and systematically studied the influence of two to 90 polyethylene glycol (PEG) lengths of PGMEs on the refolding of pig muscle lactate dehydrogenase (LDH), hen egg white lysozyme, and yeast α-glucosidase. PGMEs with longer PEG lengths such as PGME20, 50, and 90 suppressed aggregation, and increased refolding yields. Notably, PGME20 increased the LDH yield to 56.7% from 2.5% without additives. According to the refolding kinetic analysis of LDH, compared with PGME50 and 90, the refolding rate constant in PGME20 solutions remained relatively high at a broad range of concentrations because of its weaker steric hindrance of intramolecular interactions involved in folding, leading to a preference for refolding over aggregation. These findings should provide basic guidelines to identify appropriate PEG-based nonionic detergents for protein refolding.

Concatemers of Outer Membrane Protein A Take Detours in the Folding Landscape.

Outer membrane protein A (OmpA) is the most abundant protein in the outer membrane of Escherichia coli. The N-terminal domain forms an eight-stranded membrane-embedded β-barrel that is widely used as a model protein for in vitro folding into the membrane and into surfactant micelles. Under conditions that include a low surfactant concentration, OmpA can form stable higher-order structures by intermolecular association. Other β-barrel membrane proteins also associate to form noncovalently linked trimers in vivo. This inspired us to test how topological constraints imposed by intramolecular links between individual OmpA molecules affect this process. Here we report on the properties of concatemers consisting of two and three copies of the transmembrane part of OmpA. Both concatemers could be folded to a native state in surfactant micelles according to spectroscopy and electrophoretic band shifts. This native state had the same thermodynamic stability against chemical denaturation as the original OmpA. Above 1.5 M GdmCl, concatemerization increased both refolding and unfolding rates, which we attribute to entropic effects. However, below 1.5 M GdmCl, folding kinetics were 2-3 orders of magnitude slower and more complex, involving a greater degree of parallel folding steps and species that could be classified as off-pathway. Only OmpA2 could quantitatively be folded into vesicles (though to an extent lower than that of OmpA), while OmpA3 formed three species with different levels of folding. Thus, close spatial and sequential proximity of OmpA domains on the same polypeptide chain have a strong tendency to trap the protein in different misfolded states.

An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

Unique Features of Halophilic Proteins.

Proteins from moderate and extreme halophiles have unique characteristics. They are highly acidic and hydrophilic, similar to intrinsically disordered proteins. These characteristics make the halophilic proteins soluble in water and fold reversibly. In addition to reversible folding, the rate of refolding of halophilic proteins from denatured structure is generally slow, often taking several days, for example, for extremely halophilic proteins. This slow folding rate makes the halophilic proteins a novel model system for folding mechanism analysis. High solubility and reversible folding also make the halophilic proteins excellent fusion partners for soluble expression of recombinant proteins.

Effects of glycosylated (2S,4R)-hydroxyproline on the stability and assembly of collagen triple helices.

Functionalized collagen-mimetic peptides (CMPs) have been widely used in the preparation of collagen-related biomaterials. Among the reported results, the induced noncovalent interactions between the implanted functional groups or moieties were frequently the key elements to promote the self-assembly of small CMPs. In this work, we designed and synthesized a series of glycosylated CMPs in which 4-O-[β-D-galactopyranosyl]-(2S,4R)-4-hydroxyproline (Hyp(Gal)) was incorporated to explore the effects of glycosylation on the stability and assembly of collagen triple helices. Circular dichroism measurements showed that glycosylation of hydroxyproline slightly destabilized the collagen triple helices, but did not reduce their refolding rate. Compared to non-glycosylated CMPs, the incorporation of Hyp(Gal) speeded up the assembly of CMPs, indicating that this modification could assist the self-assembly of CMPs into higher-order structures, such as fibrils. O-Galactosylation of hydroxyproline imposes contrary effects on the triple helix stability and the self-assembly rate of collagen triple helices, exhibiting a piece of important and useful information for designing collagen-related biomaterials. Our finding also suggests that instead of stabilizing the triple helical conformation of CMPs, installing additional forces between CMPs could be a crucial factor to promote the assembly of CMPs into large-scale constructs.

Designing an Antibody-Based Chaperoning System through Programming the Binding and Release of the Folding Intermediate.

The protein folding pathway consists of sequential intramolecular interactions, while chaperones exert their functions either by stabilizing folding intermediates or by preventing nonspecific intermolecular interactions, which are often associated with aggregation involving exposed hydrophobic residues in folding intermediates. As chaperones do not possess specificity for individual client proteins, we designed an antibody-based chaperoning system to mimic the sequential binding and release of client proteins undergoing folding. The single-chain variable fragment of antibody (scFv) A4 binds to human muscle creatine kinase (HCK) and prevents it from aggregating. The slow dissociation of HCK from A4 resulted in delayed but eventually high-quality refolding, as reflected by the higher recovery of enzymatic activity as well as abolished aggregation. Peptide P6, a sequence in HCK involved in A4 binding, competes with HCK, promotes its dissociation from A4, and accelerates the rate of high-quality refolding. The sequential addition of A4 and P6 is essential for the chaperoning effect. The programmed binding/release method can also be applied to refold HCK from inclusion bodies. Because the association/dissociation of the folding intermediate with the antibody is highly specific, the method can be used to design tailored refolding systems and to investigate chaperoning effects on protein folding/aggregation in a sequence-specific manner.

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca2+ concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca2+-activated state of NCS proteins, little is known about the conformational dynamics of the Mg2+-bound and apo states, both of which are populated, at least transiently, at resting Ca2+ conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg2+ and in the absence of divalent ions. Under tension, the Mg2+-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca2+-bound state, the presence of Mg2+ allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.

Determining the Rate of Unfolding and Refolding of FNIII Domains by Labeling Buried Cysteine

We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in ten FNIII domains from fibronectin. This gave a comprehensive analysis of unfolding/folding kinetics of each domain, equivalent to analysis by H-D exchange (Table 1). Domains with similar stabilities like FNIII3Cys & FNIII12Cys can have very different folding/unfolding kinetics. In a previous study (JBC 286:26375-82), buried Cys in domains 2,3,11,12,6 labeled with maleimide in FN matrix fibrils. In the present study these are not obviously distinguished from 7, which did not label.