The S100 proteins make up a family of dimeric calcium binding proteins that function in response to changing calcium levels. Several S100 binding proteins have been identified; however, the exact ...biological functions of the S100 proteins are largely unknown as there are several factors which modulate their functions. To address these issues, the specificity of binding of representative members of the human S100 proteins to short N-terminal peptides of annexin I (AnI) and annexin II (AnII) was investigated under controlled experimental conditions. AnI and AnII have been shown previously to interact with S100A11 and S100A10, respectively. This provided a unique opportunity to determine their binding specificity with the other members of the human S100 protein family. It was found that AnI binds S100A6 or S100A11 while AnII binds S100A10 or S100A11. This is the first report of the interaction between S100A6 and AnI. The fact that AnI and AnII bind to selected members of the S100 protein family shows that these interactions are specific and that the mode of binding is different from that of calmodulin, as it was found not to bind AnI or AnII. From the analysis of the thermodynamics of interactions, the binding seems to be entropically driven. It was found that both AnI and AnII undergo a coil-to-helix transition upon binding to their respective binding partners. The observation that there is an overlap in functionality is not surprising due to considerable sequence homology between S100 protein family members. In fact, the functional overlap can explain previous failures of S100 knockout constructs to show any detectable changes in phenotype despite numerous implications of these proteins in important cellular processes.
Glutaminase C is a key metabolic enzyme, which is unregulated in many cancer systems and believed to play a central role in the Warburg effect, whereby cancer cells undergo changes to an altered ...metabolic profile. A long-standing hypothesis links enzymatic activity to the protein oligomeric state, hence the study of the solution behavior in general and the oligomer state in particular of glutaminase C is important for the understanding of the mechanism of protein activation and inhibition. In this report, this is extensively investigated in correlation to enzyme concentration or phosphate level, using a high-throughput microfluidic-mixing chip for the SAXS data collection, and we confirm that the oligomeric state correlates with activity. The in-depth solution behavior analysis further reveals the structural behavior of flexible regions of the protein in the dimeric, tetrameric and octameric state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C-termini of the enzyme.
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•Combination of SAXS and MD simulations can explain the molecular background for the stability studies.•Destabilizing excipients in general induce opening of the N-lobe by binding to ...specific residues in the protein.•The fully open conformation of transferrin is not seen without aggregation.
Transferrin is an attractive candidate for drug delivery due to its ability to cross the blood brain barrier. However, in order to be able to use it for therapeutic purposes, it is important to investigate how its stability depends on different formulation conditions. Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes. Lowering pH induces opening of the transferrin N-lobe, which results in a negative effect on the stability. Presence of NaCl or arginine at low pH enhances the opening and has a negative impact on the overall protein stability.
Protein-based therapeutics have become an essential part of medical treatment. They are highly specific, have high affinity and fewer off-target effects. However, stabilization of proteins is critical, time-consuming, and expensive, and it is not yet possible to predict the behavior of proteins under different conditions. The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and therefore with high potential in drug delivery. Combination of high throughput unfolding techniques and structural studies, using small angle X-ray scattering and molecular dynamic simulations, allows us to understand the behavior of transferrin on a molecular level.
β-Sheets are a common secondary structural element found in proteins. The difficulty in studying β-sheet folding and stability is that their formation is often dependent on the tertiary structural ...environment within the protein. However, the discovery of water-soluble β-hairpin peptides has allowed them to be used as model systems because they represent the smallest units of β-sheet structure independent of tertiary structural context. Trpzip4 has been used as a model β-hairpin peptide to study β-hairpin folding and stability because it is highly soluble in aqueous solutions, maintains its monomeric state, and shows reversible cooperative thermal unfolding. The previously determined thermodynamic parameters for trpzip4 thermal unfolding vary depending on the spectroscopic probe used, which questions the assumption that trpzip4 unfolds in a two-state manner. Here we provide direct calorimetric evidence that the unfolding of trpzip4 follows a two-state unfolding mode. Furthermore, the thermal unfolding of trpzip4 monitored using near- and far-UV−CD yielded thermodynamic parameters similar to those determined calorimetrically, providing additional evidence for a two-state unfolding mode.
The name of the fourth author was spelled incorrectly. (2013) Correction: Small Angle X-Ray Scattering Studies of Mitochondrial Glutaminase C Reveal Extended Flexible Regions, and Link Oligomeric ...State with Enzyme Activity.
The native reversible self-association of monoclonal antibodies has been associated with high viscosity, liquid-liquid, and liquid-solid phase separation. We investigated the native reversible ...self-association of an IgG1, which exerts this association even at low protein concentrations, in detail to gain further understanding of this phenomenon by extensive characterization of the association as a function of multiple factors, namely pH, temperature, salt concentration, and protein concentration. The nature of the self-association of the full-length IgG1 as well as the corresponding Fab and Fc fragment was studied by viz. size exclusion chromatography combined with multiangle light scattering, batch dynamic and static light scattering, analytical ultracentrifugation, small angle X-ray scattering, asymmetric flow field flow fractionation coupled with multiangle light scattering, and intrinsic fluorescence. We rationalized the self-association as a combination of hydrophobic and electrostatic interactions driven by the Fab fragments. Finally, we investigated the long-term stability of the IgG1 molecule.
There are many fluorescence-based applications that can be used to characterize molecular interactions. However, available methods often depend on site-specific labeling techniques or binding-induced ...changes in conformation or size of the probed target molecule. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformational changes. We demonstrate that in-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules, including proteins, antibodies, and nucleic acids. Thereby, the described method is not limited to a subset of molecules since even the most challenging samples of research and drug discovery projects like membrane proteins and intrinsically disordered proteins can be analyzed.
Differential scanning calorimetry can measure the heat capacity of a protein/peptide solution over a range of temperatures at constant pressure, which is used to determine the enthalpy function of ...the system. There are several experimental factors that can have a significant impact on the determined enthalpy and subsequent derived thermodynamic parameters. These factors are discussed in terms of sample and instrument preparation, as well as data collection and analysis.
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