Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Global analysis of kinetics reveals the role of secondary nucleation in recombinant spider silk self-assembly.

Recombinant spider silk proteins can be prepared in scalable fermentation processes and have been proven as sources of biomaterials for biomedical and technical applications. Nanofibrils, formed through the self-assembly of these proteins, possess unique structural and mechanical properties, serving as fundamental building blocks for the fabrication of micro- and nanostructured scaffolds. Despite significant progress in utilizing nanofibrils-based morphologies of recombinant spider silk proteins, a comprehensive understanding of the molecular mechanisms of nanofibrils self-assembly remains a challenge. Here, a detailed kinetic study of nanofibril formation from a recombinant spider silk protein eADF4(C16) in dependence on the protein concentration, seeding, and temperature is provided. For the global fitting of kinetic data obtained during the fibril formation, we utilized the online platform AmyloFit. Evaluation of the data revealed that the self-assembly mechanism of recombinant spider silk is dominated by secondary nucleation. Thermodynamic analyses show that both primary and secondary nucleations, as well as the elongation step of the eADF4(C16), are endothermic processes.

Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2.

Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.

Aggregation mechanism and branched 3D morphologies of pathological human light chain proteins under reducing conditions.

Here, we examined the aggregation mechanism and structures of the pathological human multiple myeloma light chain aggregates (hLC) after disrupting stabilizing disulfide bonds by various reducing agents. The aggregation kinetics were measured in the presence of three commonly used disulfide reducers (TCEP, DTT and glutathione), and the resulting aggregates were visualized by the combination of light and confocal/super-resolution STED microscopy. We find that aggregation kinetics can be described by two apparent macroscopic rate constants of the Finke-Watzky model related to the nucleation and the growth process. Surprisingly, the growth rate constants decreased at higher protein concentrations, which we interpret as the involvement of an aggregation active monomer particle that is successively depleted at high concentrations due to shifts in a monomer/dimer equilibrium. Seeding experiments demonstrated the specificity of the aggregates; only certain seeds accelerated the aggregation, while others eventually slowed down the aggregation. Three-dimensional visualization of the overall structures of the final aggregates at submicrometer resolution showed variable, reducer-specific branched morphologies with non-trivial fractal dimensions. Thus, the disruption of the stabilizing disulfide bonds in hLC leads to specific large, branched aggregates formed by the monomer-addition mechanism.

Protein Nanomechanics.

For a comprehensive understanding of protein function and dynamics, it is crucial to study their mechanical properties [...].

Direct observation of chemo-mechanical coupling in DnaK by single-molecule force experiments.

Protein allostery requires a communication channel for functional regulation between distal sites within a protein. In the molecular chaperone Hsp70, a two-domain enzyme, the ATP/ADP status of an N-terminal nucleotide-binding domain regulates the substrate affinity of a C-terminal substrate-binding domain. Recently available three-dimensional structures of Hsp70 in ATP/ADP states have provided deep insights into molecular pathways of allosteric signals. However, direct mechanical probing of long-range allosteric coupling between the ATP hydrolysis step and domain states is missing. Using laser optical tweezers, we examined the mechanical properties of a truncated two-domain DnaK(1-552ye) in apo/ADP/ATP- and peptide-bound states. We find that in the apo and ADP states, DnaK domains are mechanically stable and rigid. However, in the ATP state, substrate-binding domain (SBD)∗ye is mechanically destabilized as the result of interdomain docking followed by the unfolding of the α-helical lid. By observing the folding state of the SBD, we could observe the continuous ATP/ADP cycling of the enzyme in real time with a single molecule. The SBD lid closure is strictly coupled to the chemical steps of the ATP hydrolysis cycle even in the presence of peptide substrate.

Different Approaches for the Profiling of Cancer Pathway-Related Genes in Glioblastoma Cells.

Deregulation of signalling pathways that regulate cell growth, survival, metabolism, and migration can frequently lead to the progression of cancer. Brain tumours are a large group of malignancies characterised by inter- and intratumoral heterogeneity, with glioblastoma (GBM) being the most aggressive and fatal. The present study aimed to characterise the expression of cancer pathway-related genes (n = 84) in glial tumour cell lines (A172, SW1088, and T98G). The transcriptomic data obtained by the qRT-PCR method were compared to different control groups, and the most appropriate control for subsequent interpretation of the obtained results was chosen. We analysed three widely used control groups (non-glioma cells) in glioblastoma research: Human Dermal Fibroblasts (HDFa), Normal Human Astrocytes (NHA), and commercially available mRNAs extracted from healthy human brain tissues (hRNA). The gene expression profiles of individual glioblastoma cell lines may vary due to the selection of a different control group to correlate with. Moreover, we present the original multicriterial decision making (MCDM) for the possible characterization of gene expression profiles. We observed deregulation of 75 genes out of 78 tested in the A172 cell line, while T98G and SW1088 cells exhibited changes in 72 genes. By comparing the delta cycle threshold value of the tumour groups to the mean value of the three controls, only changes in the expression of 26 genes belonging to the following pathways were identified: angiogenesis FGF2; apoptosis APAF1, CFLAR, XIAP; cellular senescence BM1, ETS2, IGFBP5, IGFBP7, SOD1, TBX2; DNA damage and repair ERCC5, PPP1R15A; epithelial to mesenchymal transition SNAI3, SOX10; hypoxia ADM, ARNT, LDHA; metabolism ATP5A1, COX5A, CPT2, PFKL, UQCRFS1; telomeres and telomerase PINX1, TINF2, TNKS, and TNKS2. We identified a human astrocyte cell line and normal human brain tissue as the appropriate control group for an in vitro model, despite the small sample size. A different method of assessing gene expression levels produced the same disparities, highlighting the need for caution when interpreting the accuracy of tumorigenesis markers.

Allosteric Inter-Domain Contacts in Bacterial Hsp70 Are Located in Regions That Avoid Insertion and Deletion Events.

Heat shock proteins 70 (Hsp70) are chaperones consisting of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD), the latter of which binds protein clients. After ATP binds to the NBD, the SBD α/ß subdomains' shared interface opens, and the open SBD docks to the NBD. Such allosteric effects are stabilized by the newly formed NBD-SBD interdomain contacts. In this paper, we examined how such an opening and formation of subdomain interfaces is affected during the evolution of Hsp70. In particular, insertion and deletion events (indels) can be highly disruptive for the mechanical events since such changes introduce a collective shift in the pairing interactions at communicating interfaces. Based on a multiple sequence alignment analysis of data collected from Swiss-Prot/UniProt database, we find several indel-free regions (IFR) in Hsp70. The two largest IFRs are located in interdomain regions that participate in allosteric structural changes. We speculate that the reason why the indels have a lower likelihood of occurrence in these regions is that indel events in these regions cause dysfunction in the protein due to perturbations of the mechanical balance. Thus, the development of functional allosteric machines requires including in the rational design a concept of the balance between structural elements.

Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria.

14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer-monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.

Single-molecule mechanical studies of chaperones and their clients.

Single-molecule force spectroscopy provides access to the mechanics of biomolecules. Recently, magnetic and laser optical tweezers were applied in the studies of chaperones and their interaction with protein clients. Various aspects of the chaperone-client interactions can be revealed based on the mechanical probing strategies. First, when a chaperone is probed under load, one can examine the inner workings of the chaperone while it interacts with and works on the client protein. Second, when protein clients are probed under load, the action of chaperones on folding clients can be studied in great detail. Such client folding studies have given direct access to observing actions of chaperones in real-time, like foldase, unfoldase, and holdase activity. In this review, we introduce the various single molecule mechanical techniques and summarize recent single molecule mechanical studies on heat shock proteins, chaperone-mediated folding on the ribosome, SNARE folding, and studies of chaperones involved in the folding of membrane proteins. An outlook on significant future developments is given.

Interpretation of Single-Molecule Force Experiments on Proteins Using Normal Mode Analysis.

Single-molecule force spectroscopy experiments allow protein folding and unfolding to be explored using mechanical force. Probably the most informative technique for interpreting the results of these experiments at the structural level makes use of steered molecular dynamics (MD) simulations, which can explicitly model the protein under load. Unfortunately, this technique is computationally expensive for many of the most interesting biological molecules. Here, we find that normal mode analysis (NMA), a significantly cheaper technique from a computational perspective, allows at least some of the insights provided by MD simulation to be gathered. We apply this technique to three non-homologous proteins that were previously studied by force spectroscopy: T4 lysozyme (T4L), Hsp70 and the glucocorticoid receptor domain (GCR). The NMA results for T4L and Hsp70 are compared with steered MD simulations conducted previously, and we find that we can recover the main results. For the GCR, which did not undergo MD simulation, our approach identifies substructures that correlate with experimentally identified unfolding intermediates. Overall, we find that NMA can make a valuable addition to the analysis toolkit for the structural analysis of single-molecule force experiments on proteins.

Assessing the Viscoelasticity of Photopolymer Nanowires Using a Three-Parameter Solid Model for Bending Recovery Motion.

Photopolymer nanowires prepared by two-photon polymerization direct laser writing (TPP-DLW) are the building blocks of many microstructure systems. These nanowires possess viscoelastic characteristics that define their deformations under applied forces when operated in a dynamic regime. A simple mechanical model was previously used to describe the bending recovery motion of deflected nanowire cantilevers in Newtonian liquids. The inverse problem is targeted in this work; the experimental observations are used to determine the nanowire physical characteristics. Most importantly, based on the linear three-parameter solid model, we derive explicit formulas to calculate the viscoelastic material parameters. It is shown that the effective elastic modulus of the studied nanowires is two orders of magnitude lower than measured for the bulk material. Additionally, we report on a notable effect of the surrounding aqueous glucose solution on the elasticity and the intrinsic viscosity of the studied nanowires made of Ormocomp.

Classifying Residues in Mechanically Stable and Unstable Substructures Based on a Protein Sequence: The Case Study of the DnaK Hsp70 Chaperone.

Artificial proteins can be constructed from stable substructures, whose stability is encoded in their protein sequence. Identifying stable protein substructures experimentally is the only available option at the moment because no suitable method exists to extract this information from a protein sequence. In previous research, we examined the mechanics of E. coli Hsp70 and found four mechanically stable (S class) and three unstable substructures (U class). Of the total 603 residues in the folded domains of Hsp70, 234 residues belong to one of four mechanically stable substructures, and 369 residues belong to one of three unstable substructures. Here our goal is to develop a machine learning model to categorize Hsp70 residues using sequence information. We applied three supervised methods: logistic regression (LR), random forest, and support vector machine. The LR method showed the highest accuracy, 0.925, to predict the correct class of a particular residue only when context-dependent physico-chemical features were included. The cross-validation of the LR model yielded a prediction accuracy of 0.879 and revealed that most of the misclassified residues lie at the borders between substructures. We foresee machine learning models being used to identify stable substructures as candidates for building blocks to engineer new proteins.

SlyD Accelerates trans-to-cis Prolyl Isomerization in a Mechanosignaling Protein under Load.

Prolyl isomerization is recognized as one of the key regulatory mechanisms, which plays a crucial role in cell signaling, ion channel gating, phage virus infection, and molecular timing. This isomerization is usually slow but often accelerated by an enzyme, called peptidyl-prolyl isomerase (PPIase). In the current project, we investigate using single-molecule force spectroscopy (SMFS) the impact of a bacterial PPIase, SlyD, on the cis-trans isomerization of the proline 2225 (P2225) in an isolated 20th domain of a cytoskeletal mechanosensing protein filamin-A (FlnA20). To explore the FlnA20-PPIase interaction, we have used multiple SMFS modes, like constant velocity, constant distance, and jumping trap experiments. In our previous study, we reported the unique nature of the P2225, which is conserved in all naturally occurring filamins and can slowly (minutes) interconvert between cis-trans isomers, in absence of any PPIase. Our current results show a staggering 25-fold acceleration of the trans-to-cis isomerization rate in the presence of saturating SlyD concentration (7.25 µM) compared to the unenzymatic condition. A SlyD concentration-dependent depletion of the trans isomeric lifetime was also observed. Additionally, we observed that SlyD stabilizes the cis-isomer in the native state of FlnA20 by ∼2 kBT. This is the first single-molecule observation of the cis-trans isomerization catalysis by a PPIase in a mechanosensing protein.

Salt-dependent passive adsorption of IgG1κ-type monoclonal antibodies on hydrophobic microparticles.

Understanding how antibodies adsorb on solid surfaces is essential for developing effective approaches to control this process. In this study, passive adsorptions on the hydrophobic solid surface of a polystyrene microparticle (MP) of two highly similar IgG1 κ-type monoclonal antibodies (mAbs), rituximab, and trastuzumab, were examined in the presence of Hofmeister salts. Except of kosmotropic salts, the screening of electrostatic interactions using salts reduces the passive adsorption of mAbs on MP. To better understand the ion-specific adsorption process, salt-dependent Langmuir isotherm parameters were obtained and correlated for two mAbs. We find that while their maximum adsorption capacities to MPs are highly correlated (r > 0.9), the salt-dependent profiles of adsorption binding constants, Kobs, differ substantially. For rituximab, Kobs increases >10-fold in an ion-specific manner; for trastuzumab, Kobs remains constant. We conclude that even minor sequence variations among the mAbs can affect the adsorption, as well as the molecular forces attracting proteins to a solid surface. This difference might originate from the heterogeneous orientation of the adsorbed mAbs.

Nanoimpact in Plants: Lessons from the Transcriptome.

Transcriptomics studies are available to evaluate the potential toxicity of nanomaterials in plants, and many highlight their effect on stress-responsive genes. However, a comparative analysis of overall expression changes suggests a low impact on the transcriptome. Environmental challenges like pathogens, saline, or drought stress induce stronger transcriptional responses than nanoparticles. Clearly, plants did not have the chance to evolve specific gene regulation in response to novel nanomaterials; but they use common regulatory circuits with other stress responses. A shared effect with abiotic stress is the inhibition of genes for root development and pathogen response. Other works are reviewed here, which also converge on these results.

Entropy-Based Strategies for Rapid Pre-Processing and Classification of Time Series Data from Single-Molecule Force Experiments.

Recent advances in single-molecule science have revealed an astonishing number of details on the microscopic states of molecules, which in turn defined the need for simple, automated processing of numerous time-series data. In particular, large datasets of time series of single protein molecules have been obtained using laser optical tweezers. In this system, each molecular state has a separate time series with a relatively uneven composition from the point of view-point of local descriptive statistics. In the past, uncertain data quality and heterogeneity of molecular states were biased to the human experience. Because the data processing information is not directly transferable to the black-box-framework for an efficient classification, a rapid evaluation of a large number of time series samples simultaneously measured may constitute a serious obstacle. To solve this particular problem, we have implemented a supervised learning method that combines local entropic models with the global Lehmer average. We find that the methodological combination is suitable to perform a fast and simple categorization, which enables rapid pre-processing of the data with minimal optimization and user interventions.

A kinetic coupling between protein unfolding and aggregation controls time-dependent solubility of the human myeloma antibody light chain.

Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In general, time-dependent changes in protein solubility are complex kinetically driven processes that often involve a triggering event that consists of a protein unfolding/misfolding followed by the assembling of aggregation-competent protein species. In this study, we have examined the relation between stability and time-dependent solubility of the recombinant human antibody light chain, hLC, which was found to form renal tubular casts in the multiple myeloma patient. To analyze the aggregation quantitatively, the hLC stability and protein solubility assays were performed in vitro at elevated temperatures. A differential acceleration of the processes at high temperatures enabled us to dissect observed kinetics of irreversible hLC unfolding and aggregation. We find that for hLC these processes have different molecularity and activation energy barriers. While the irreversible unfolding of hLC is a unimolecular step with a substantial activation energy barrier of 260 kJ/mol, the aggregation is rate-limited by the bimolecular reaction, which is characterized by a lower activation energy barrier of 40 kJ/mol. By the combination of experimental assays at different temperatures, different protein concentrations and kinetic modeling using ordinary differential equations, we were able to extrapolate time-dependent protein solubility to temperatures where both unfolding and aggregation processes are strongly kinetically coupled. Our study enables mechanism-based evaluation and interpretation of different physico-chemical factors contributing to the hLC unfolding and aggregation and their effect on the formation of extracellular protein deposits.

Ion-Specific Protein/Water Interface Determines the Hofmeister Effect on the Kinetic Stability of Glucose Oxidase.

Homodimeric glucose oxidase (GOX) from Aspergillus niger is a prominent enzyme used for a number of applications in biotechnology and clinical diagnostics. For robust and long-term functional applications of GOX, the stability of the protein is of utmost importance. In vitro, GOX is irreversibly inactivated over time by a mechanism that is poorly understood, and hence, it presents a significant drawback for the development of strategies to stabilize the enzyme. We show that the nonequilibrium stability of GOX is fully described by a one-step conformational unfolding kinetics. To explore the strategies for improving GOX nonequilibrium stability, the effect of salts of the Hofmeister series is examined using microcalorimetry. We obtain activation energies Ea and inactivation temperatures Tk (at which the irreversible step is 1.0 min-1) as a function of the salt types and concentrations. Based on the analysis by the extended Langmuir model, we find that at high salt concentrations (>1 M) the Hofmeister effect on inactivation temperature is determined by the universal ion-specific effect on the protein/water interface, which apparently does not depend significantly on a particular amino-acid sequence and 3D protein structure. Our findings identify protein/water interfacial tension as a critical physicochemical attribute of excipients that is crucial for increasing enzyme kinetic stability.

An Active, Ligand-Responsive Pulling Geometry Reports on Internal Signaling between Subdomains of the DnaK Nucleotide-Binding Domain in Single-Molecule Mechanical Experiments.

Single-molecule mechanical experiments have proven to be ideal tools for probing the energetics and mechanics of large proteins and domains. In this paper, we investigate the nucleotide-dependent unfolding mechanics of the nucleotide-binding domain (NBD) of the Hsp70 chaperone DnaK. The NBD binds ADP or ATP in the binding cleft formed by lobe I and lobe II, which consists of two subdomains each. When force is applied to the termini of the NBD, the observed unfolding forces are independent of the nucleotide state. In contrast, when force is applied across the nucleotide-binding pocket, the unfolding forces report specifically on the nucleotide-phosphate state. In this active, ligand-responsive pulling geometry, we observed a bifurcation of the unfolding pathway; the pathway proceeds either through a cooperative "coupled pathway" or through a noncooperative "uncoupled pathway". The partitioning between individual unfolding pathways can be effectively tuned by mutation or by the nucleotide exchange factor GrpE, i.e., by the factors affecting the strength of the lobe I-lobe II interactions within the native NBD. These experiments provide important insight into the molecular origin of the internal signaling between the subdomains of the nucleotide-binding domain of Hsp70 proteins and how signals are efficiently transferred inside the protein molecule.