Prediction of the Stability of Protein Substructures Using AI/ML Techniques.

This chapter explores the innovative application of machine learning techniques to understand and predict the stability of protein substructures. Accurately identifying stable substructures within proteins necessitates incorporating the local context, crucial for elucidating the roles of supersecondary structures. This approach emphasizes the importance of contextual information in understanding the stability and functionality of protein regions, thereby providing a more comprehensive view of protein mechanics and interactions.

On the production of singlet oxygen by the isoalloxazine ring in free and protein-bound flavin cofactors.

Flavin cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), as a part of flavoenzymes play a critical role in the catalysis of multiple reactions predominantly of a redox nature. Question arises why nature developed two very similar cofactors with an identical functional part - isoalloxazine ring. We believe that an answer is related to the fact that the isoalloxazine ring belongs to endogenous photosensitizers able to produce reactive and potentially harmful singlet oxygen, O, with high efficiency, Φ ∼ 0.

On the feasibility of time-resolved X-ray powder diffraction of macromolecules using laser-driven ultrafast X-ray sources.

With the emergence of ultrafast X-ray sources, interest in following fast processes in small molecules and macromolecules has increased. Most of the current research into ultrafast structural dynamics of macromolecules uses X-ray free-electron lasers. In parallel, small-scale laboratory-based laser-driven ultrafast X-ray sources are emerging.

Design and fabrication of 3D-printed crystallization plates for probing microcrystals in an external electric field.

X-ray crystallography is an established tool to probe the structure of macromolecules with atomic resolution. Compared with alternative techniques such as single-particle cryo-electron microscopy and micro-electron diffraction, X-ray crystallography is uniquely suited to room-temperature studies and for obtaining a detailed picture of macromolecules subjected to an external electric field (EEF). The impact of an EEF on proteins has been extensively explored through single-crystal X-ray crystallography, which works well with larger high-quality protein crystals.

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 near the prolyl isomerase active site.

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.

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 ( 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.

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.

Protein Nanomechanics.

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

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.

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.

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 ( = 84) in glial tumour cell lines (A172, SW1088, and T98G).

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/β 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.

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 K values of the dimer-monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.

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.

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.

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 Hsp70 and found four mechanically stable (S class) and three unstable substructures (U class).

SlyD Accelerates -to- 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 isomerization of the proline 2225 (P2225) in an isolated 20th domain of a cytoskeletal mechanosensing protein filamin-A (FlnA20).

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.

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.

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.

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.

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

Homodimeric glucose oxidase (GOX) from 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.

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.

Stable Substructures in Proteins and How to Find Them Using Single-Molecule Force Spectroscopy.

Three-dimensional structures of proteins are a source of fascination for scientists, due to the beauty of their sequence-encoded architectures and their highly diverse range of functions. These functions include acting as powerful catalysts, signal receptors, and versatile molecular motors as well as being building blocks for macroscopic structures, thus defining the shape of multicellular organisms. How protein structure is organized and assembled at the sub-nanometer scale is of great current interest.