Nickel-ion substituted hydroxyapatite matrices for metal-affinity chromatographic purification of recombinant proteins.

Hydroxyapatite (HAp) is a calcium phosphate ceramic, widely used as a matrix for protein chromatography. The crystal structure of HAp is amenable to a wide range of substitutions, thus allowing for the alteration of its properties. In this study, nickel-ion substituted HAp (NiSHAp) was synthesized using a wet-precipitation method, followed by spray drying. This resulted in the structural incorporation of nickel ions within well-defined microspheres, which were suitable for chromatographic applications. The chromatographic experiments were conducted with NiSHAp and compared with spray-dried hydroxyapatite (SHAp) matrices. Protein purification experiments were conducted using refolded recombinant L-asparaginase (L-Asp), which was produced as inclusion bodies in Escherichia coli. The results showed that NiSHAp effectively adsorbed L-Asp, which was selectively eluted using a phosphate buffer, surpassing the efficiency of imidazole-based elution. In contrast, SHAp showed weaker binding and lower selectivity. The significance of this study lies in developing a scalable NiSHAp matrix for protein purification, especially for large-scale applications. The NiSHAp matrix offers a cost-effective alternative to commercial immobilized metal affinity chromatography (IMAC) adsorbents, especially for purifying His-tagged proteins. This innovative approach exhibits the advantages of mixed-mode chromatography by combining the properties of hydroxyapatite and IMAC in a single matrix, with the potential of improved industrial-scale protein purification.

Impact of Charge State on Characterization of Large Middle-Down Sized Peptides by Tandem Mass Spectrometry.

Fragmentation trends of large peptides were characterized by five activation methods, including HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD. Sequence coverages and scores were assessed based on charge site, peptide sequence, and peptide size. The effect of charge state and peptide size on sequence coverage was explored for a Glu-C digest of E. coli ribosomal proteins, and linear regression analysis of the collection of peptides indicated that HCD, ETD, and EThcD have a higher dependence charge state than 193 and 213 nm UV. Four model peptides, neuromedin, glucagon, galanin, and amyloid ß, were characterized in greater detail based on charge site analysis and showed a charge state dependence on sequence coverage for collision and electron-based activation methods.

Nanosecond Transient IR Spectroscopy of Halorhodopsin in Living Cells.

The ability to track minute changes of a single amino acid residue in a cellular environment is causing a paradigm shift in the attempt to fully understand the responses of biomolecules that are highly sensitive to their environment. Detecting early protein dynamics in living cells is crucial to understanding their mechanisms, such as those of photosynthetic proteins. Here, we elucidate the light response of the microbial chloride pump NmHR from the marine bacterium Nonlabens marinus, located in the membrane of living Escherichia coli cells, using nanosecond time-resolved UV/vis and IR absorption spectroscopy over the time range from nanoseconds to seconds. Transient structural changes of the retinal cofactor and the surrounding apoprotein are recorded using light-induced time-resolved UV/vis and IR difference spectroscopy. Of particular note, we have resolved the kinetics of the transient deprotonation of a single cysteine residue during the photocycle of NmHR out of the manifold of molecular vibrations of the cells. These findings are of high general relevance, given the successful development of optogenetic tools from photoreceptors to interfere with enzymatic and neuronal pathways in living organisms using light pulses as a noninvasive trigger.

A universal method for surface-based binding assays by preparing immobilized ß2-adrenergic receptor stationary phase using solid binding peptide as a fusion tag.

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize ß2-adrenergic receptor (ß2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4-1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged ß2-adrenoceptor (ß2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, ß2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on ß2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M-1 respectively, which were two orders of magnitude higher than those on the Halo-ß2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.

Oriented immobilization of nanobodies using SpyCatcher/SpyTag significantly enhances the capacity of affinity chromatography.

The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.

Molecular insights into capsular polysaccharide secretion.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.

FSHing for DNA Damage: Key Features of MutY Detection of 8-Oxoguanine:Adenine Mismatches.

Base excision repair (BER) enzymes are genomic superheroes that stealthily and accurately identify and remove chemically modified DNA bases. DNA base modifications erode the informational content of DNA and underlie many disease phenotypes, most conspicuously, cancer. The "OG" of oxidative base damage, 8-oxo-7,8-dihydroguanine (OG), is particularly insidious due to its miscoding ability that leads to the formation of rare, pro-mutagenic OG:A mismatches. Thwarting mutagenesis relies on the capture of OG:A mismatches prior to DNA replication and removal of the mis-inserted adenine by MutY glycosylases to initiate BER. The threat of OG and the importance of its repair are underscored by the association between inherited dysfunctional variants of the MutY human homologue (MUTYH) and colorectal cancer, known as MUTYH-associated polyposis (MAP). Our functional studies of the two founder MUTYH variants revealed that both have compromised activity and a reduced affinity for OG:A mismatches. Indeed, these studies underscored the challenge of the recognition of OG:A mismatches that are only subtly structurally different than T:A base pairs. Since the original discovery of MAP, many MUTYH variants have been reported, with most considered to be "variants of uncertain significance." To reveal features associated with damage recognition and adenine excision by MutY and MUTYH, we have developed a multipronged chemical biology approach combining enzyme kinetics, X-ray crystallography, single-molecule visualization, and cellular repair assays. In this review, we highlight recent work in our laboratory where we defined MutY structure-activity relationship (SAR) studies using synthetic analogs of OG and A in cellular and in vitro assays. Our studies revealed the 2-amino group of OG as the key distinguishing feature of OG:A mismatches. Indeed, the unique position of the 2-amino group in the major groove of OGsyn:Aanti mismatches provides a means for its rapid detection among a large excess of highly abundant and structurally similar canonical base pairs. Furthermore, site-directed mutagenesis and structural analysis showed that a conserved C-terminal domain ß-hairpin "FSH'' loop is critical for OG recognition with the "His" serving as the lesion detector. Notably, MUTYH variants located within and near the FSH loop have been associated with different forms of cancer. Uncovering the role(s) of this loop in lesion recognition provided a detailed understanding of the search and repair process of MutY. Such insights are also useful to identify mutational hotspots and pathogenic variants, which may improve the ability of physicians to diagnose the likelihood of disease onset and prognosis. The critical importance of the "FSH" loop in lesion detection suggests that it may serve as a unique locus for targeting probes or inhibitors of MutY/MUTYH to provide new chemical biology tools and avenues for therapeutic development.

Exploring the dynamics and structure of PpiB in living Escherichia coli cells using electron paramagnetic resonance spectroscopy.

The combined effects of the cellular environment on proteins led to the definition of a fifth level of protein structural organization termed quinary structure. To explore the implication of potential quinary structure for globular proteins, we studied the dynamics and conformations of Escherichia coli (E. coli) peptidyl-prolyl cis/trans isomerase B (PpiB) in E. coli cells. PpiB plays a major role in maturation and regulation of folded proteins by catalyzing the cis/trans isomerization of the proline imidic peptide bond. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) and nitroxide spin labels. In addition to using standard spin labeling approaches with genetically engineered cysteines, we incorporated an unnatural amino acid to achieve Gd(III)-nitroxide orthogonal labeling. We probed PpiB's residue-specific dynamics by X-band continuous wave EPR at ambient temperatures and its structure by double electron-electron resonance (DEER) on frozen samples. PpiB was delivered to E. coli cells by electroporation. We report a significant decrease in the dynamics induced by the cellular environment for two chosen labeling positions. These changes could not be reproduced by adding crowding agents and cell extracts. Concomitantly, we report a broadening of the distance distribution in E. coli, determined by Gd(III)-Gd(III) DEER measurements, as compared with solution and human HeLa cells. This suggests an increase in the number of PpiB conformations present in E. coli cells, possibly due to interactions with other cell components, which also contributes to the reduction in mobility and suggests the presence of a quinary structure.

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE.

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.

Revealing Allosteric Mechanism of Amino Acid Binding Proteins from Open to Closed State.

Amino acid binding proteins (AABPs) undergo significant conformational closure in the periplasmic space of Gram-negative bacteria, tightly binding specific amino acid substrates and then initiating transmembrane transport of nutrients. Nevertheless, the possible closure mechanisms after substrate binding, especially long-range signaling, remain unknown. Taking three typical AABPs-glutamine binding protein (GlnBP), histidine binding protein (HisJ) and lysine/arginine/ornithine binding protein (LAOBP) in Escherichia coli (E. coli)-as research subjects, a series of theoretical studies including sequence alignment, Gaussian network model (GNM), anisotropic network model (ANM), conventional molecular dynamics (cMD) and neural relational inference molecular dynamics (NRI-MD) simulations were carried out. Sequence alignment showed that GlnBP, HisJ and LAOBP have high structural similarity. According to the results of the GNM and ANM, AABPs' Index Finger and Thumb domains exhibit closed motion tendencies that contribute to substrate capture and stable binding. Based on cMD trajectories, the Index Finger domain, especially the I-Loop region, exhibits high molecular flexibility, with residues 11 and 117 both being potentially key residues for receptor-ligand recognition and initiation of receptor allostery. Finally, the signaling pathway of AABPs' conformational closure was revealed by NRI-MD training and trajectory reconstruction. This work not only provides a complete picture of AABPs' recognition mechanism and possible conformational closure, but also aids subsequent structure-based design of small-molecule oncology drugs.

Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity.

The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCATI) and several EcCATI:ligand-bound complexes. EcCATI readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap EcCATI and Chl in a ternary, ligand-bound state (EcCATI:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from EcCATI:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to EcCATI even as protein unfolding occurs. Gas-phase binding constants further suggest that EcCATI binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to EcCATI when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of EcCATI.

Effect of Tryptophan Metabolites on Cell Damage Revealed by Bacteria-Cell Interactions in Hydrogel Microspheres.

This work presented an alternative approach for studying bacteria-cell interactions in three-dimensional (3D) hydrogel microspheres formed by the cross-linking reaction of alginate and calcium-ethylenediaminetetraacetic acid (EDTA-Ca) produced in a microfluidic chip. During the co-culture process of hepatocytes (HepG2) and Escherichia coli (E. coli) 25922, we concluded that the content change of tryptophan metabolites detected via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was related to the cell damage level and the change of interleukin (IL-22) detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was related to the ways of co-cultivation. Compared to the two-dimensional (2D) adherent cell culture process in a Petri dish (2D), the co-culture process of HepG2 and E. coli 25922 in hydrogel microspheres indicated more information about metabolism such as the appearance of indole-3-propionic acid (IPA) and possibly IL-22. The method provides a new perspective to investigate the bacteria-cell interaction and it could be a promising tool in the study of gut microbiota and human health.

Structural basis of Rho-dependent transcription termination.

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli1-4. Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho's function.

Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli: Dissecting and Mitigating Redox Heterogeneity.

Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG1-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG1 CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.

AAA+ protease-adaptor structures reveal altered conformations and ring specialization.

ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here we present high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze the protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated, tucked away from the channel and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies show additional structures and functions for key AAA+ elements. Pore-loop tucking may be used broadly by AAA+ unfoldases, for example, during enzyme pausing/unloading.

Remodelling the surface of thioredoxin from Escherichia coli by grafting an iron-binding site from the CyaY protein family.

In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(III) and Al(III). The interaction of the grafted TRX with iron(III) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(III) per TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(III), a Fe(III) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical.

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein.

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4'-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C8-CoA. Furthermore, our 1H saturation transfer difference and 31P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved 71RGG73 loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

In vitro eradication of abasic site-mediated DNA-peptide/protein cross-links by Escherichia coli long-patch base excision repair.

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA-protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5'-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5'-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2-36.1 kDa) DPCs is less efficient than that of an AP-peptide10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.

Multimodal approaches for the improvement of the cellular folding of a recombinant iron regulatory protein in E. coli.

BACKGROUND: During the recombinant protein expression, most heterologous proteins expressed in E. coli cell factories are generated as insoluble and inactive aggregates, which prohibit E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The presence of folding modulators such as exogenous molecular chaperones or osmolytes, as well as process variables such as incubation temperature, inducer concentrations, growth media are all important for cellular folding and are investigated in this study. This study also investigated how the cell's stress response system activates and protects the proteins from aggregation. RESULTS: The cells exposed to osmolytes plus a pre-induction heat shock showed a substantial increase in recombinant aconitase activity when combined with modulation of process conditions. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells. CONCLUSIONS: The recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase production during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression.

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP), and Stoichiometry of GroEL-GroES Complexes.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm ∼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.