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Antibodies (Basel, Switzerland) Oct 2023The poly-reactivity of antibodies is defined as their binding to specific antigens as well as to related proteins and also to unrelated targets. Poly-reactivity can... (Review)
Review
The poly-reactivity of antibodies is defined as their binding to specific antigens as well as to related proteins and also to unrelated targets. Poly-reactivity can occur in individual molecules of natural serum antibodies, likely due to their conformation flexibility, and, for therapeutic antibodies, it plays a critical role in their clinical development. On the one hand, it can enhance their binding to target antigens and cognate receptors, but, on the other hand, it may lead to a loss of antibody function by binding to off-target proteins. Notably, poly-reactivity has been observed in antibodies subjected to treatments with dissociating, destabilizing or denaturing agents, in particular acidic pH, a common step in the therapeutic antibody production process involving the elution of Protein-A bound antibodies and viral clearance using low pH buffers. Additionally, poly-reactivity can emerge during the affinity maturation in the immune system, such as the germinal center. This review delves into the underlying potential causes of poly-reactivity, highlighting the importance of conformational flexibility, which can be further augmented by the acid denaturation of antibodies and the introduction of arginine mutations into the complementary regions of antibody-variable domains. The focus is placed on a particular antibody's acid conformation, meticulously characterized through circular dichroism, differential scanning calorimetry, and sedimentation velocity analyses. By gaining a deeper understanding of these mechanisms, we aim to shed light on the complexities of antibody poly-reactivity and its implications for therapeutic applications.
PubMed: 37873861
DOI: 10.3390/antib12040064 -
ACS Nano Jul 2022The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge...
The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge of single-cell proteomics, single-molecule technologies are being developed and improved. Most approaches, to date, rely on the analysis of polypeptides, resulting from digested proteins, either in solution or immobilized on a surface. Nanopore biosensing is an emerging single-molecule technique that circumvents surface immobilization and is optimally suited for the analysis of long biopolymers, as has already been shown for DNA sequencing. However, proteins, unlike DNA molecules, are not uniformly charged and harbor complex tertiary structures. Consequently, the ability of nanopores to analyze unfolded full-length proteins has remained elusive. Here, we evaluate the use of heat denaturation and the anionic surfactant sodium dodecyl sulfate (SDS) to facilitate electrokinetic nanopore sensing of unfolded proteins. Specifically, we characterize the voltage dependence translocation dynamics of a wide molecular weight range of proteins (from 14 to 130 kDa) through sub-5 nm solid-state nanopores, using a SDS concentration below the critical micelle concentration. Our results suggest that proteins' translocation dynamics are significantly slower than expected, presumably due to the smaller nanopore diameters used in our study and the role of the electroosmotic force opposing the translocation direction. This allows us to distinguish among the proteins of different molecular weights based on their dwell time and electrical charge deficit. Given the simplicity of the protein denaturation assay and circumvention of the tailor-made necessities for sensing protein of different folded sizes, shapes, and charges, this approach can facilitate the development of a whole proteome identification technique.
Topics: Nanopores; Proteome; DNA; Electroosmosis; Nanotechnology
PubMed: 35785960
DOI: 10.1021/acsnano.2c05391 -
Biosensors Dec 2022We report non-contact laser-based Brillouin light-scattering (BLS) spectroscopy measurements of the viscoelastic properties of hyperthermally radiofrequency (RF)-heated...
We report non-contact laser-based Brillouin light-scattering (BLS) spectroscopy measurements of the viscoelastic properties of hyperthermally radiofrequency (RF)-heated and ablated bovine liver and chicken flesh tissues with embedded gold nanoparticles (AuNPs). The spatial lateral profile of the local surface temperature in the flesh samples during their hyperthermia was measured through optical backscattering reflectometry (OBR) using Mg−silica-NP-doped sensing fibers distributed with an RF applicator and correlated with viscoelastic variations in heat-affected and ablated tissues. Substantial changes in the tissue stiffness after heating and ablation were directly related to their heat-induced structural modifications. The main proteins responsible for muscle elasticity were denatured and irreversibly aggregated during the RF ablation. At T > 100 °C, the proteins constituting the flesh further shrank and became disorganized, leading to substantial plastic deformation of biotissues. Their uniform destruction with larger thermal lesions and a more viscoelastic network was attained via AuNP-mediated RF hyperthermal ablation. The results demonstrated here pave the way for simultaneous real-time hybrid optical sensing of viscoelasticity and local temperature in biotissues during their denaturation and gelation during hyperthermia for future applications that involve mechanical- and thermal-property-controlled theranostics.
Topics: Animals; Cattle; Hot Temperature; Gold; Hyperthermia, Induced; Metal Nanoparticles; Temperature
PubMed: 36671844
DOI: 10.3390/bios13010008 -
Molecular Therapy. Nucleic Acids Sep 2020Preservation of denatured dermis exerts promotive functions in wound healing and improves the appearance and function of skin. Angiogenesis is crucial for wound healing...
Preservation of denatured dermis exerts promotive functions in wound healing and improves the appearance and function of skin. Angiogenesis is crucial for wound healing during burn injury. However, the potential molecular mechanism of angiogenesis in the recovery after burn injury remains to be elucidated. Herein, RNA chromatin immunoprecipitation (ChIP) sequencing analysis revealed upregulation of long intergenic non-coding RNA 00174 (linc00174) in the post-burn tissues. linc00174 overexpression promoted angiogenic activities of human umbilical vein endothelial cells (HUVECs) in the heat-denatured cell model, characterized by the promotion of cell proliferation, migration, and tube formation. Mechanistically, linc00174 directly bound to enhancer of zeste homolog 2 (EZH2), thus stimulating the protein level of trimethylation at lysine 27 of histone H3 (H3K27me3). Moreover, inhibition of EZH2 resulted in downregulation of ZNF24 and Runx1, as well as a decline of vascular endothelial growth factor A (VEGFA). Furthermore, EZH2 modulated epigenetic repression of ZNF24 and Runx1 through the promoter of H3K27me3. Additionally, ZNF24 and Runx1 both functioned as transcriptional inhibitors of VEGFA. Taken together, these findings uncover that linc00174 epigenetically inhibits ZNF24 and Runx1 expression through binding to EZH2, thus attenuating the suppression of VEGFA, contributing to the facilitation of angiogenesis during the recovery of heat-denatured endothelial cells.
PubMed: 32805486
DOI: 10.1016/j.omtn.2020.07.010 -
Biotechnology and Bioengineering May 2021Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA...
Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and four proteins, three of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature as low as 75°C with a treatment duration of 15 min can effectively inactivate CoV. The degree of CoV heat inactivation greatly depends on the length of heat treatment time and the temperature applied. With the goal of finding whether sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple fluidic system that can measure sub-second heat inactivation of CoV. The system is composed of a stainless-steel capillary immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can flow at various speeds. Flowing virus solution at different speeds, along with temperature control and monitoring system, allows the virus to be exposed to the desired temperature and treatment durations with high accuracy. Using mouse hepatitis virus, a betacoronavirus, as a model CoV system, we identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log reduction in viral titer (starting titer: 5 × 10 PFU/ml), and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated (>6 Log reduction).
Topics: Betacoronavirus; Hot Temperature; Murine hepatitis virus; Viral Plaque Assay; Virus Inactivation
PubMed: 33615450
DOI: 10.1002/bit.27720 -
Molecules (Basel, Switzerland) Jun 2022Functional amyloid is produced by many organisms but is particularly well understood in bacteria, where proteins such as CsgA () and FapC () are assembled as functional... (Review)
Review
Functional amyloid is produced by many organisms but is particularly well understood in bacteria, where proteins such as CsgA () and FapC () are assembled as functional bacterial amyloid (FuBA) on the cell surface in a carefully optimized process. Besides a host of helper proteins, FuBA formation is aided by multiple imperfect repeats which stabilize amyloid and streamline the aggregation mechanism to a fast-track assembly dominated by primary nucleation. These repeats, which are found in variable numbers in Pseudomonas, are most likely the structural core of the fibrils, though we still lack experimental data to determine whether the repeats give rise to β-helix structures via stacked β-hairpins (highly likely for CsgA) or more complicated arrangements (possibly the case for FapC). The response of FuBA fibrillation to denaturants suggests that nucleation and elongation involve equal amounts of folding, but protein chaperones preferentially target nucleation for effective inhibition. Smart peptides can be designed based on these imperfect repeats and modified with various flanking sequences to divert aggregation to less stable structures, leading to a reduction in biofilm formation. Small molecules such as EGCG can also divert FuBA to less organized structures, such as partially-folded oligomeric species, with the same detrimental effect on biofilm. Finally, the strong tendency of FuBA to self-assemble can lead to the formation of very regular two-dimensional amyloid films on structured surfaces such as graphite, which strongly implies future use in biosensors or other nanobiomaterials. In summary, the properties of functional amyloid are a much-needed corrective to the unfortunate association of amyloid with neurodegenerative disease and a testimony to nature's ability to get the best out of a protein fold.
Topics: Amyloid; Amyloidogenic Proteins; Bacterial Proteins; Biofilms; Escherichia coli; Humans; Neurodegenerative Diseases; Pseudomonas
PubMed: 35807329
DOI: 10.3390/molecules27134080 -
Biochemistry Aug 2019Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone activities. It is a U-shaped protein with an structural organization in...
Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone activities. It is a U-shaped protein with an structural organization in which the and domains have CGHC active sites, the and domains are involved with substrate binding, and is a flexible linker. PDI exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions. To determine whether this unfolding-refolding cycle is a substrate-induced process or an intrinsic physical property of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal denaturation. PDI exhibited remarkable conformational resilience that is linked to its redox status. In the reduced state, PDI exhibited a 54 °C unfolding transition temperature () and regained 85% of its native structure after nearly complete thermal denaturation. Oxidized PDI had a lower of 48-50 °C and regained 70% of its native conformation after 75% denaturation. Both reduced PDI and oxidized PDI were functional after refolding from these denatured states. Additional studies documented increased stability of a PDI construct lacking the domain and decreased thermal stability of a construct lacking the domain. Furthermore, oxidation of the domain limited the ability of PDI to refold. The stability and conformational resilience of PDI are thus linked to both redox-dependent and domain-specific effects. These findings document previously unrecognized properties of PDI and provide insight into the physical foundation of its biological function.
Topics: Cholera Toxin; Circular Dichroism; Humans; Oxidation-Reduction; Protein Conformation; Protein Disulfide-Isomerases; Protein Folding; Protein Stability
PubMed: 31393106
DOI: 10.1021/acs.biochem.9b00405 -
Scientific Reports Sep 2022The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications....
The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR-Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9-DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR-Cas9 technology.
Topics: CRISPR-Associated Protein 9; DNA; Electrophoretic Mobility Shift Assay; RNA, Guide, CRISPR-Cas Systems; Temperature
PubMed: 36085316
DOI: 10.1038/s41598-022-19485-x -
ACS Omega Jan 2022Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive...
Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive understanding of the mechanisms underlying encapsulin self-assembly, disassembly, and reassembly is lacking. Here, we characterize the disassembly/reassembly properties of three encapsulin nanocages that possess different structural architectures: = 1 (24 nm), = 3 (32 nm), and = 4 (42 nm). Using spectroscopic techniques and electron microscopy, encapsulin architectures were found to exhibit varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature. While all three encapsulins showed the capacity to reassemble following GuHCl-induced disassembly (within 75 min), only the smallest = 1 nanocage reassembled after disassembly in basic pH (within 15 min). Furthermore, atomic force microscopy revealed that all encapsulins showed a significant loss of structural integrity after undergoing sequential disassembly/reassembly steps. These findings provide insights into encapsulins' disassembly/reassembly dynamics, thus informing their future design, modification, and application.
PubMed: 35036749
DOI: 10.1021/acsomega.1c05472 -
Scientific Reports Mar 2022The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in...
The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.
Topics: COVID-19 Nucleic Acid Testing; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Real-Time Polymerase Chain Reaction; SARS-CoV-2; Saliva; Specimen Handling
PubMed: 35273232
DOI: 10.1038/s41598-022-07871-4