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Chemical cross-linking of a variety of green fluorescent proteins as F?rster resonance energy transfer donors for Yukon orange fluorescent protein: A project-based undergraduate laboratory experience
摘要： F?rster resonance energy transfer (FRET) is the basis for many techniques used in biomedical research. Due to its wide use in molecular sensing, FRET is commonly introduced in many biology, chemistry, and physics courses. While FRET is of great importance in the biophysical sciences, the complexity and dif?culty of constructing FRET experiments has resulted in limited usage in undergraduate laboratory settings. Here, we present a practical undergraduate laboratory experiment for teaching FRET using a diverse set of green-emitting ?uorescent proteins (FPs) as donors for a cross-linked Yukon orange FP. This laboratory experiment enables students to make the connection of basic lab procedures to real world applications and can be applied to molecular biology, biochemistry, physical chemistry, and biophysical laboratory courses.
关键词： Upper-division undergraduate,orange ?uorescent protein,?uorescence lifetimes,proteins,green ?uorescent protein,biochemistry,TCSPC,?uorescence spectroscopy
更新于2025-09-10 09:29:36
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[Progress in Molecular Biology and Translational Science] Volume 160 || Fluorescent Proteins as Sensors for Cellular Behavior in Mice
摘要： Imaging of cancer cells in mice expressing fluorescent proteins has allowed the real-time tracing of cancer growth and metastasis and determination of efficacy of candidate antitumor and antimetastatic agents, especially in mouse orthotopic models. The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm can visualize the nuclear–cytoplasmic dynamics of cancer cells in vivo, including mitosis, apoptosis, cell-cycle position, and differential behavior of nucleus and cytoplasm that occurs during cancer cell deformation, migration, and extravasation. Recent applications of the technology described here include linking fluorescent proteins with cell cycle-specific proteins such that the cells change color from red to green as they transit from G1 to S phases. Any in vivo process can be imaged using fluorescent proteins, allowing molecular biology to advance from in vitro studies to studying molecular processes in the living animal.
关键词： cancer imaging,metastasis,RFP,in vivo imaging,GFP,fluorescent proteins
更新于2025-09-10 09:29:36
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Small-Angle Neutron Scattering Reveals Energy Landscape for Rhodopsin Photoactivation
摘要： Knowledge of the activation principles for G-protein-coupled receptors (GPCRs) is critical to development of new pharmaceuticals. Rhodopsin is the archetype for the largest GPCR family, yet the changes in protein dynamics that trigger signaling are not fully understood. Here we show that rhodopsin can be investigated by small-angle neutron scattering (SANS) in fully protiated detergent micelles under contrast matching to resolve light-induced changes in the protein structure. In SANS studies of membrane proteins, the zwitterionic detergent [(Cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS) is advantageous because of the low contrast difference between the hydrophobic core and hydrophilic head groups as compared to alkyl glycoside detergents. Combining SANS results with quasielastic neutron scattering (QENS) reveals how changes in volumetric protein shape are coupled (slaved) to the aqueous solvent. Upon light exposure rhodopsin is swollen by penetration of water into the protein core, allowing interactions with effector proteins in the visual signaling mechanism.
关键词： Detergent,Neutron Scattering,Hydration,GPCR,Protein Dynamics,Vision,Membrane Proteins,Energy landscape,Slaving,Rhodopsin
更新于2025-09-10 09:29:36
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Nanoscale tweezers for single-cell biopsies
摘要： Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional ‘snapshot’ of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10–20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.
关键词： single-cell biopsies,nanotweezers,proteins,DNA,mitochondrion,dielectrophoretic trapping
更新于2025-09-10 09:29:36
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Enhanced Fluorescent Protein Activity in Polymer Scaffold-Stabilized Phospholipid Nanoshells Using Neutral Redox Initiator Polymerization Conditions
摘要： Phospholipid nanoshells, for example, liposomes, provide a versatile enabling platform for the development of nanometer-sized biosensors and molecular delivery systems. Utilization of phospholipid nanoshells is limited by the inherent instability in complex biological environments, where the phospholipid nanoshell may disassemble and degrade, thus releasing the contents and destroying sensor function. Polymer sca?old stabilization (PSS), wherein the phospholipid nanoshells are prepared by partitioning reactive monomers into the lipid bilayer lamella followed by radical polymerization, has emerged to increase phospholipid nanoshell stability. In this work, we investigated the e?ects of three di?erent radical initiator conditions to fabricate stable PSS-phospholipid nanoshells yet retain the activity of encapsulated model ?uorescent sensor proteins. To identify nondestructive initiation conditions, UV photoinitiation, neutral redox initiation, and thermal initiation were investigated as a function of PSS-phospholipid nanoshell stabilization and ?uorescence emission intensity of enhanced green ?uorescent protein (eGFP) and tandem dimer Tomato (td-Tomato). All three initiator approaches yielded comparably stable PSS-phospholipid nanoshells, although slight variations in PSS-phospholipid nanoshell size were observed, ranging from ca. 140 nm for unstabilized phospholipid nanoshells to 300?500 nm for PSS-phospholipid nanoshells. Fluorescence emission intensity of encapsulated eGFP was completely attenuated under thermal initiation (0% vs control), moderately attenuated under UV photoinitiation (40 ± 4% vs control), and una?ected by neutral redox initiation (97 ± 3% vs control). Fluorescence emission intensity of encapsulated td-Tomato was signi?cantly attenuated under thermal initiation (13 ± 3% vs control), moderately attenuated UV photoinitiation (64 ± 5% vs control), and una?ected by neutral redox initiation (98% ± 4% vs control). Therefore, the neutral redox initiation method provides a signi?cant advancement toward the preparation of protein-functionalized PSS-phospholipid nanoshells. These results should help to guide future applications and designs of biosensor platforms using PSS-phospholipid nanoshells and other polymer systems employing protein transducers.
关键词： Phospholipid nanoshells,td-Tomato,Neutral redox initiation,UV photoinitiation,Radical initiator conditions,eGFP,Polymer sca?old stabilization,Thermal initiation,Fluorescent sensor proteins
更新于2025-09-09 09:28:46
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A Flow Cytometric Study of ER Stress and Autophagy
摘要： The mechanistic link between ER stress, autophagy, and resultant cell death was investigated by the use of drugs Thapsigargin (Tg) and Chloroquine (CQ) with prior induction and or blockade of autophagy and apoptosis which modulated the ER stress response and resultant form of cell death. All these biological processes can be measured flow cytometrically allowing the determination of the type of cell death, G1 cell cycle arrest, cell cycle dependent measurement of ER stress transducer PERK, misfolded proteins, reticulophagy, and autophagy marker LC3B. Jurkat cells after Tg or CQ treatment became necrotic and apoptotic, showed G1 cell cycle arrest, autophagy, and ER stress. Prior induction of autophagy before ER stress increased levels of necrotic and apoptotic cell death. Autophagy was further up-regulated, while PERK was reduced or abrogated. CQ showed reduced levels of misfolded proteins and reticulophagy, while Tg showed no change in misfolded protein levels but increased reticulophagy and thus displayed more ER stress. Prior blockade of apoptosis before induction of ER stress resulted in cell survival except with high Tg levels which induced necrosis. Autophagy was up-regulated with modulation of PERK and reticulophagy levels with an abrogation of the misfolded protein response. Blockade of apoptosis with induction of autophagy before ER stress showed death by necrosis with high dose drugs and cell survival with low doses of drugs. CQ induced reduced levels G1 cell cycle arrest while it was maintained with Tg. Autophagy was also maintained with reduced levels of ER stress. These data demonstrates a profound link between the processes of ER stress, autophagy, and the resultant form of cell death all of which can be modulated depending upon the sequence and concentration of drugs employed.
关键词： misfolded proteins,autophagy,PERK,ER stress,reticulophagy
更新于2025-09-09 09:28:46
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Targeted Feature Extraction in MALDI Mass Spectrometry Imaging to Discriminate Proteomic Profiles of Breast and Ovarian Cancer
摘要： Purpose: To develop a mass spectrometry imaging (MSI) based workflow for extracting m/z values related to putative protein biomarkers and using these for reliable tumor classification. Experimental design: Given a list of putative breast and ovarian cancer biomarker proteins, we extracted a set of related m/z values from heterogeneous MSI datasets derived from formalin-fixed paraffin-embedded tissue material. Based on these features, a linear discriminant analysis classification model was trained to discriminate the two tumor types. Results: We show that the discriminative power of classification models based on the extracted features is increased compared to the automatic training approach, especially when classifiers are applied to spectral data acquired under different conditions (instrument, preparation, laboratory). Conclusions and clinical relevance: We obtained robust classification models not confounded by technical variation between MSI measurements. This supports the assumption that the classification of the respective tumor types is based on biological rather than technical differences, and that the selected features are related to the proteomic profiles of the tumor types under consideration.
关键词： feature extraction,tumor typing,MALDI MSI,tissue classification,biomarker proteins
更新于2025-09-09 09:28:46
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Chiral Analysis || Raman Optical Activity
摘要： Raman optical activity (ROA) has many advantages over conventional Raman spectroscopy for investigation of chiral molecules, in general, and biological molecules, in particular, on account of their homochirality. Since it is a manifestation of vibrational optical activity [1], being complementary to vibrational circular dichroism (VCD), ROA is more sensitive to the 3D structures of chiral molecules through its dependence on their absolute handedness, which has led to its development as an incisive probe of the structure and behavior of biomolecules in aqueous solution. For example, ROA spectra obtained from proteins can provide detailed information on the secondary and tertiary structures adopted by polypeptide backbones. Additionally, ROA can probe the tautomers of side chains. In common with other vibrational spectroscopies, there is no restriction on the size of molecules, or in their type, that can be investigated by ROA, broadening its application to unfolded proteins, viruses, and carbohydrates.
关键词： vibrational circular dichroism,proteins,Raman optical activity,aqueous solution,carbohydrates,vibrational optical activity,biological molecules,biomolecules,viruses,chiral molecules
更新于2025-09-09 09:28:46
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moxMaple3: a Photoswitchable Fluorescent Protein for PALM and Protein Highlighting in Oxidizing Cellular Environments
摘要： The ability of fluorescent proteins (FPs) to fold robustly is fundamental to the autocatalytic formation of the chromophore. While the importance of the tertiary protein structure is well appreciated, the impact of individual amino acid mutations for FPs is often not intuitive and requires direct testing. In this study, we describe the engineering of a monomeric photoswitchable FP, moxMaple3, for use in oxidizing cellular environments, especially the eukaryotic secretory pathway. Surprisingly, a point mutation to replace a cysteine substantially improved the yield of correctly folded FP capable of chromophore formation, regardless of cellular environment. The improved folding of moxMaple3 increases the fraction of visibly tagged fusion proteins, as well as FP performance in PALM super-resolution microscopy, and thus makes moxMaple3 a robust monomeric FP choice for PALM and optical highlighting applications.
关键词： protein highlighting,oxidizing cellular environments,photoswitchable,PALM,fluorescent proteins
更新于2025-09-09 09:28:46
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Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization
摘要： The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important but poorly resolved are the forces present during a membrane protein’s initial unfolding, where the most native set of interactions are present. We developed a high-precision, atomic force microscopy assay to study the initial unfolding of bacteriorhodopsin. We discovered rapid near-equilibrium folding between the first three unfolding states that corresponded to the unfolding of 5 and 8 amino-acids respectively when using a cantilever optimized for 2-μs resolution. Interestingly, the third of these states was retinal stabilized and previously undetected despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for >1 min. We expect that this ability to measure the rapid and reversible dynamics in the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native-like conditions.
关键词： protein folding,single molecule force spectroscopy,site-specific bioconjugation,membrane proteins,atomic force microscopy
更新于2025-09-04 15:30:14