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Molecules (Basel, Switzerland) Jun 2021The inhibition of the protein function for therapeutic applications remains challenging despite progress these past years. While the targeting application of molecularly...
The inhibition of the protein function for therapeutic applications remains challenging despite progress these past years. While the targeting application of molecularly imprinted polymer are in their infancy, no use was ever made of their magnetic hyperthermia properties to damage proteins when they are coupled to magnetic nanoparticles. Therefore, we have developed a facile and effective method to synthesize magnetic molecularly imprinted polymer nanoparticles using the green fluorescent protein (GFP) as the template, a bulk imprinting of proteins combined with a grafting approach onto maghemite nanoparticles. The hybrid material exhibits very high adsorption capacities and very strong affinity constants towards GFP. We show that the heat generated locally upon alternative magnetic field is responsible of the decrease of fluorescence intensity.
Topics: Green Fluorescent Proteins; Magnetic Iron Oxide Nanoparticles; Molecularly Imprinted Polymers; Protein Denaturation
PubMed: 34210027
DOI: 10.3390/molecules26133980 -
Cellular and Molecular Life Sciences :... Oct 2007To be, or not to be--that is the question not only for Hamlet in Shakespeare's drama but also for a protein associated with molecular chaperones. While long viewed... (Review)
Review
To be, or not to be--that is the question not only for Hamlet in Shakespeare's drama but also for a protein associated with molecular chaperones. While long viewed exclusively as cellular folding factors, molecular chaperones recently emerged as active participants in protein degradation. This places chaperones at the center of a life or death decision during protein triage. Here we highlight molecular mechanisms that underlie chaperone action at the folding/degradation interface in mammalian cells. We discuss the importance of chaperone-assisted degradation for the regulation of cellular processes and its emerging role as a target for therapeutic intervention in cancer and amyloid diseases.
Topics: Animals; Apoptosis; Humans; Molecular Chaperones; Neoplasms; Neurodegenerative Diseases; Protein Denaturation; Proteins; Ubiquitin
PubMed: 17565442
DOI: 10.1007/s00018-007-7188-6 -
Journal of the American Chemical Society Aug 2006Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold...
Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below -25 degrees C. Comparison of cold-induced structural transitions in a variety of reverse micelle-buffer systems indicate that protein-surfactant interactions are negligible and allow the direct observation of the multistate cold-induced unfolding of the protein.
Topics: Cold Temperature; Models, Molecular; Protein Conformation; Protein Denaturation; Ubiquitin
PubMed: 16910639
DOI: 10.1021/ja0628654 -
Analytical Chemistry Apr 2023Stability of high-concentration protein formulations is considered a major challenge in current biopharmaceutical development. In this work, we introduce laser-based...
Stability of high-concentration protein formulations is considered a major challenge in current biopharmaceutical development. In this work, we introduce laser-based mid-infrared (IR) spectroscopy as a versatile technique to study the effect of protein concentration and presence of sugars on the thermal denaturation of the model protein bovine serum albumin (BSA). Many analytical techniques struggle to characterize the complex structural transition that occurs during protein denaturation. To this end, a commercially available laser-based mid-IR spectrometer equipped with a customized flow cell was employed to record IR spectra of BSA in the temperature range of 25-85 °C. The temperature perturbation induces a conformational change from a native α-helical to an intermolecular β-sheet secondary structure in BSA. Systematic investigation of the concentration dependence of the α-β transition temperature between 30 and 90 mg mL shows a trend of decreasing denaturation temperatures at higher BSA concentrations. In-depth chemometric analysis by a multivariate curve resolution-alternating least squares (MCR-ALS) analysis of the spectra, suggested the formation of not one but two intermediates in the denaturation of BSA. Subsequently, the impact of sugars on denaturation temperatures was investigated, revealing both stabilizing (trehalose, sucrose, and mannose) and destabilizing (sucralose) effects, illustrating the applicability of this method as an investigative tool for stabilizers. These results highlight the potential and versatility of laser-based IR spectroscopy for analysis of protein stability at high concentrations and varying conditions.
Topics: Spectrophotometry, Infrared; Serum Albumin, Bovine; Sugars; Protein Denaturation; Lasers; Spectroscopy, Fourier Transform Infrared
PubMed: 37010404
DOI: 10.1021/acs.analchem.3c00489 -
Journal of the American Chemical Society Dec 2022Many studies, in which proteins have been unfolded by the action of a variety of physical or chemical agents, have led to the definition of a folded versus an unfolded... (Review)
Review
Many studies, in which proteins have been unfolded by the action of a variety of physical or chemical agents, have led to the definition of a folded versus an unfolded state and to the question of what is the nature of the unfolded state. The unstructured nature of this state could suggest that "the" unfolded state is a unique entity which holds true for all kinds of unfolding processes. This assumption has to be questioned because the unfolding processes under different stress conditions are dictated by entirely different mechanisms. As a consequence, it can be easily understood that the final state, generically referred to as "the unfolded state", can be completely different for each of the unfolding processes. The present review examines recent data on the characteristics of the unfolded states emerging from experiments under different conditions, focusing specific attention to the level of compaction of the unfolded species.
Topics: Protein Folding; Proteins; Protein Denaturation
PubMed: 36450361
DOI: 10.1021/jacs.2c07696 -
European Biophysics Journal : EBJ Jan 2018Protein thermodynamic stability is intricately linked to cellular function, and altered stability can lead to dysfunction and disease. The linear extrapolation model...
Protein thermodynamic stability is intricately linked to cellular function, and altered stability can lead to dysfunction and disease. The linear extrapolation model (LEM) is commonly used to obtain protein unfolding free energies ([Formula: see text]) by extrapolation of solvent denaturation data to zero denaturant concentration. However, for some proteins, different denaturants result in non-coincident LEM-derived [Formula: see text] values, raising questions about the inherent assumption that the obtained [Formula: see text] values are intrinsic to the protein. Here, we used single-molecule FRET measurements to better understand such discrepancies by directly probing changes in the dimensions of the protein G B1 domain (GB1), a well-studied protein folding model, upon urea and guanidine hydrochloride denaturation. A comparison of the results for the two denaturants suggests denaturant-specific structural energetics in the GB1 denatured ensemble, revealing a role of the denatured state in the variable thermodynamic behavior of proteins.
Topics: Bacterial Proteins; Fluorescence Resonance Energy Transfer; Guanidine; Protein Denaturation; Protein Domains; Thermodynamics; Urea
PubMed: 29080139
DOI: 10.1007/s00249-017-1260-4 -
The Biochemical Journal May 19651. Soluble calf-skin collagen has been denatured thermally between 37 degrees and 60 degrees and the component proteins have been separated on carboxymethylcellulose. 2....
1. Soluble calf-skin collagen has been denatured thermally between 37 degrees and 60 degrees and the component proteins have been separated on carboxymethylcellulose. 2. Four main fractions have been separated; alpha and beta (in the nomenclature in common usage) and two other fractions. (The alpha and beta components are complex owing to the presence of alpha(1), alpha(2), beta(1) and beta(2) parts). 3. Fractions 3 and 4 undergo rapid denaturation between 39 degrees and 40 degrees whereafter fraction 4 remains virtually unchanged even at 60 degrees . 4. That portion of fraction 4 which remains at 60 degrees is thought to be identical with the fraction designated gamma by other workers, this fraction being composed of three alpha-chains in covalent linkage (such bonds are alkali-labile). 5. The equilibrium between alpha, beta and fractions 3 and 4 is apparently reversible since acid-soluble collagen after denaturation at 45 degrees or 60 degrees followed by cooling to 0 degrees for 30min. was found to contain only fraction 4 when chromatographed at 37 degrees .
Topics: Acids; Animals; Cattle; Chemical Phenomena; Chemistry, Physical; Chromatography; Collagen; Hot Temperature; Methylcellulose; Protein Denaturation; Research; Skin
PubMed: 14340083
DOI: 10.1042/bj0950350 -
Journal of the American Chemical Society May 2007Recent experimental work on fast protein folding brings about an intriguing paradox. Microsecond-folding proteins are supposed to fold near or at the folding speed limit...
Recent experimental work on fast protein folding brings about an intriguing paradox. Microsecond-folding proteins are supposed to fold near or at the folding speed limit (downhill folding), but yet their folding behavior seems to comply with classical two-state analyses, which imply the crossing of high free energy barriers. However, close inspection of chemical and thermal denaturation kinetic experiments in fast-folding proteins reveals systematic deviations from two-state behavior. Using a simple one-dimensional free energy surface approach we find that such deviations are indeed diagnostic of marginal folding barriers. Furthermore, the quantitative analysis of available fast-kinetic data indicates that many microsecond-folding proteins fold downhill in native conditions. All of these proteins are then promising candidates for an atom-by-atom analysis of protein folding using nuclear magnetic resonance.1 We also find that the diffusion coefficient for protein folding is strongly temperature dependent, corresponding to an activation energy of approximately 1 kJ.mol-1 per protein residue. As a consequence, the folding speed limit at room temperature is about an order of magnitude slower than the approximately 1 micros estimates from high-temperature T-jump experiments. Our analysis is quantitatively consistent with the available thermodynamic and kinetic data on slow two-state folding proteins and provides a straightforward explanation for the apparent fast-folding paradox.
Topics: Algorithms; Computer Simulation; Energy Transfer; Entropy; Kinetics; Models, Chemical; Protein Denaturation; Protein Folding; Surface Properties; Temperature; Thermodynamics
PubMed: 17419630
DOI: 10.1021/ja0689740 -
International Journal of Hyperthermia :... May 2006Hyperthermia results in protein unfolding that, if not properly chaperoned by Heat Shock Proteins (HSP), can lead to irreversible and toxic protein aggregates. Elevating... (Review)
Review
Hyperthermia results in protein unfolding that, if not properly chaperoned by Heat Shock Proteins (HSP), can lead to irreversible and toxic protein aggregates. Elevating HSP prior to heating makes cells thermotolerant. Hyperthermia also can enhance the sensitivity of cells to radiation and drugs. This sensitization to drugs or radiation is not directly related to altered HSP expression. However, altering HSP expression before heat and radiation or drug treatment will affect the extent of thermal sensitization because the HSP will attenuate the heat-induced protein damage that is responsible for radiation- or drug-sensitization. For thermal radiosensitization, nuclear protein damage is considered to be responsible for hyperthermic effects on DNA repair, in particular base excision repair. Hyperthermic drug sensitization can be seen for a number of anti-cancer drugs, especially of alkylating agents. Synergy between heat and drugs may arise from multiple events such as heat damage to ABC transporters (drug accumulation), intra-cellular drug detoxification pathways and repair of drug-induced DNA adducts. This may be why cells with acquired drug resistance (often multi-factorial) can be made responsive to drugs again by combining the drug treatment with heat.
Topics: Apoptosis; Combined Modality Therapy; Drug Therapy; Heat-Shock Proteins; Humans; Hyperthermia, Induced; Neoplasms; Nuclear Proteins; Protein Denaturation; Radiation Tolerance; Radiotherapy
PubMed: 16754338
DOI: 10.1080/02656730500532028 -
Protein Science : a Publication of the... Oct 2019Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large-scale industrial organizations. The link between ligand...
Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large-scale industrial organizations. The link between ligand affinity (K ) and ΔTm is understood for reversible denaturation; however, there is a gap in our understanding of how to quantitatively interpret ΔTm for the many proteins that irreversibly denature. To better understand the origin, and extent of applicability, of a K to ΔTm correlate, we define equations relating K and ΔTm for irreversible protein unfolding, which we test with computational models and experimental data. These results suggest a general relationship exists between K and ΔTm for irreversible denaturation.
Topics: Ligands; Models, Molecular; Protein Denaturation; Proteins; Temperature
PubMed: 31361943
DOI: 10.1002/pro.3701