Fluorescein: A Photo-CIDNP Sensitizer Enabling Hypersensitive NMR Data Collection in Liquids at Low Micromolar Concentration

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is a powerful approach for sensitivity enhancement in NMR spectroscopy. In liquids, intermolecular photo-CIDNP depends on the transient bimolecular reaction between photoexcited dye and sample of interest. Hence the extent of polarization is sample-concentration dependent. This study introduces fluorescein (FL) as a photo-CIDNP dye whose performance is exquisitely tailored to data collection at extremely low sample concentrations. The photo-CIDNP resonance intensities of tryptophan in the presence of either FL or FMN (i.e., the routinely employed flavin mononucleotide photosensitizer) in the liquid state show that FL yields superior sensitivity and enables rapid data collection down to an unprecedented 1 μM concentration. This result was achieved on a conventional spectrometer operating at 14.1 T and equipped with a room-temperature probe (i.e., noncryogenic). Kinetic simulations show that the excellent behavior of FL arises from its long excited-state triplet lifetime and superior photostability relative to conventional photo-CIDNP sensitizers

Extending the Experimentally Accessible Range of Spin Dipole–Dipole Spectral Densities for Protein–Cosolute Interactions by Temperature-Dependent Solvent Paramagnetic Relaxation Enhancement Measurements

Longitudinal (Γ1) and transverse (Γ2) solvent paramagnetic relaxation enhancement (sPRE) yields field-dependent information in the form of spectral densities that provides unique information related to cosolute–protein interactions and electrostatics. A typical protein sPRE data set can only sample a few points on the spectral density curve, J(ω), within a narrow frequency window (500 MHz to ∼1 GHz). However, complex interactions and dynamics of paramagnetic cosolutes around a protein make it difficult to directly interpret the few experimentally accessible points of J(ω). In this paper, we show that it is possible to significantly extend the experimentally accessible frequency range (corresponding to a range from ∼270 MHz to 1.8 GHz) by acquiring a series of sPRE experiments at different temperatures. This approach is based on the scaling property of J(ω) originally proposed by Melchior and Fries for small molecules. Here, we demonstrate that the same scaling property also holds for geometrically far more complex systems such as proteins. Using the extended spectral densities derived from the scaling property as the reference dataset, we demonstrate that our previous approach that makes use of a non-Lorentzian Ansatz spectral density function to fit only J(0) and one to two J(ω) points allows one to obtain accurate values for the concentration-normalized equilibrium average of the electron–proton interspin separation ⟨r–6⟩norm and the correlation time τC, which provide quantitative information on the energetics and timescale, respectively, of local cosolute–protein interactions. We also show that effective near-surface potentials, ϕENS, obtained from ⟨r–6⟩norm provide a reliable and quantitative measure of intermolecular interactions including electrostatics, while ϕENS values obtained from only Γ1 or Γ2 sPRE rates can have significant artifacts as a consequence of potential variations and changes in the diffusive properties of the cosolute around the protein surface. Finally, we discuss the experimental feasibility and limitations of extracting the high-frequency limit of J(ω) that is related to ⟨r–8⟩norm and report on the extremely local intermolecular potential

Theory and Applications of Nitroxide-based Paramagnetic Cosolutes for Probing Intermolecular and Electrostatic Interactions on Protein Surfaces

Solvent paramagnetic relaxation enhancement (sPRE) arising from nitroxide-based cosolutes has recently been used to provide an atomic view of cosolute-induced protein denaturation and to characterize residue-specific effective near-surface electrostatic potentials (ϕENS). Here, we explore distinct properties of the sPRE arising from nitroxide-based cosolutes and provide new insights into the interpretation of the sPRE and sPRE-derived ϕENS. We show that: (a) the longitudinal sPRE rate Γ1 is heavily dependent on spectrometer field and viscosity, while the transverse sPRE rate Γ2 is much less so; (b) the spectral density J(0) is proportional to the inverse of the relative translational diffusion constant and is related to the quantity ⟨r–4⟩norm, a concentration-normalized equilibrium average of the electron–proton interspin separation; and (c) attractive intermolecular interactions result in a shortening of the residue-specific effective correlation time for the electron–proton vector. We discuss four different approaches for evaluating ϕENS based on Γ2, J(0), Γ1, or ⟨r–6⟩norm. The latter is evaluated from the magnetic field dependence of Γ1 in conjunction with Γ2. Long-range interactions dominate J(0) and Γ2, while, at high magnetic fields, the contribution of short-range interactions becomes significant for J(ω) and hence Γ1; the four ϕENS quantities enable one to probe both long- and short-range electrostatic interactions. The experimental ϕENS potentials were evaluated using three model protein systems, two folded (ubiquitin and native drkN SH3) and one intrinsically disordered (unfolded state of drkN SH3), in relation to theoretical ϕENS potentials calculated from atomic coordinates using the Poisson-Boltzmann theory with either a r–6 or r–4 dependence

A “Steady-State” Relaxation Dispersion Nuclear Magnetic Resonance Experiment for Studies of Chemical Exchange in Degenerate ¹H Transitions of Methyl Groups

Degenerate spin-systems consisting of magnetically equivalent nuclear spins, such as a 1H3 spin-system in selectively 13CH3-labeled proteins, present considerable challenges for the design of Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion NMR experiments to characterize chemical exchange on the micro-to-millisecond time-scale. Several approaches have been previously proposed for the elimination of deleterious artifacts observed in methyl 1H CPMG relaxation dispersion profiles obtained for (13C)1H3 groups. We describe an alternative, experimentally simple solution and design a “steady-state” methyl 1H CPMG scheme, where 90° or acute-angle (1H radiofrequency pulses are applied after each CPMG echo in-phase with methyl 1H magnetization, resulting in the establishment of a “steady-state” for effective rates of magnetization decay. A simple computational procedure for quantitative analysis of the “steady-state” CPMG relaxation dispersion profiles is developed. The “steady-state” CPMG methodology is applied to two protein systems where exchange between major and minor species occurs in different regimes on the chemical shift time-scale

DataSheet_4_Case report: Novel NIPBL-BEND2 fusion gene identified in osteoblastoma-like phosphaturic mesenchymal tumor of the fibula.docx

Phosphaturic mesenchymal tumor (PMT) is a rare tumor that secretes fibroblast growth factor 23 (FGF23) and causes hypophosphatemia and tumor-induced osteomalacia (TIO). Fusion genes FN1-FGFR1 and FN1-FGF1 have been detected in some PMTs, but the pathogenesis of PMTs without these fusion genes remains unclear. Here, we report a 12-year-old boy with persistent muscle weakness and gait disturbance. Roentgenographic examination revealed a radiolucent lesion with endosteal scalloping in the left fibula, while his serum level of FGF23 was markedly increased. Combined with simple X-ray findings of other body parts, we suspected that TIO was caused by PMT, and resected the tumor. After resection, the serum level of FGF23 started to decrease immediately and normalized within 3 hours after resection, with this being earlier than normalization of the serum phosphorus level. In RNA sequencing, FN1-FGFR1 and FN1-FGF1 were not detected, but a novel NIPBL-BEND2 fusion gene was identified. When we forcedly expressed this fusion gene in HEK293T cells and MG63 cells, cell proliferation was enhanced in both cell lines. Furthermore, Gene set enrichment analysis of HEK293T cells showed significant upregulation of MYC-target genes. Our results suggest that this novel NIPBL-BEND2 fusion gene promotes cell proliferation possibly via the MYC pathway and might be one of the etiologies of PMTs other than FN1-FGFR1 or FN1-FGF1.</p

DataSheet_1_Case report: Novel NIPBL-BEND2 fusion gene identified in osteoblastoma-like phosphaturic mesenchymal tumor of the fibula.xlsx

Phosphaturic mesenchymal tumor (PMT) is a rare tumor that secretes fibroblast growth factor 23 (FGF23) and causes hypophosphatemia and tumor-induced osteomalacia (TIO). Fusion genes FN1-FGFR1 and FN1-FGF1 have been detected in some PMTs, but the pathogenesis of PMTs without these fusion genes remains unclear. Here, we report a 12-year-old boy with persistent muscle weakness and gait disturbance. Roentgenographic examination revealed a radiolucent lesion with endosteal scalloping in the left fibula, while his serum level of FGF23 was markedly increased. Combined with simple X-ray findings of other body parts, we suspected that TIO was caused by PMT, and resected the tumor. After resection, the serum level of FGF23 started to decrease immediately and normalized within 3 hours after resection, with this being earlier than normalization of the serum phosphorus level. In RNA sequencing, FN1-FGFR1 and FN1-FGF1 were not detected, but a novel NIPBL-BEND2 fusion gene was identified. When we forcedly expressed this fusion gene in HEK293T cells and MG63 cells, cell proliferation was enhanced in both cell lines. Furthermore, Gene set enrichment analysis of HEK293T cells showed significant upregulation of MYC-target genes. Our results suggest that this novel NIPBL-BEND2 fusion gene promotes cell proliferation possibly via the MYC pathway and might be one of the etiologies of PMTs other than FN1-FGFR1 or FN1-FGF1.</p

DataSheet_3_Case report: Novel NIPBL-BEND2 fusion gene identified in osteoblastoma-like phosphaturic mesenchymal tumor of the fibula.xlsx

Phosphaturic mesenchymal tumor (PMT) is a rare tumor that secretes fibroblast growth factor 23 (FGF23) and causes hypophosphatemia and tumor-induced osteomalacia (TIO). Fusion genes FN1-FGFR1 and FN1-FGF1 have been detected in some PMTs, but the pathogenesis of PMTs without these fusion genes remains unclear. Here, we report a 12-year-old boy with persistent muscle weakness and gait disturbance. Roentgenographic examination revealed a radiolucent lesion with endosteal scalloping in the left fibula, while his serum level of FGF23 was markedly increased. Combined with simple X-ray findings of other body parts, we suspected that TIO was caused by PMT, and resected the tumor. After resection, the serum level of FGF23 started to decrease immediately and normalized within 3 hours after resection, with this being earlier than normalization of the serum phosphorus level. In RNA sequencing, FN1-FGFR1 and FN1-FGF1 were not detected, but a novel NIPBL-BEND2 fusion gene was identified. When we forcedly expressed this fusion gene in HEK293T cells and MG63 cells, cell proliferation was enhanced in both cell lines. Furthermore, Gene set enrichment analysis of HEK293T cells showed significant upregulation of MYC-target genes. Our results suggest that this novel NIPBL-BEND2 fusion gene promotes cell proliferation possibly via the MYC pathway and might be one of the etiologies of PMTs other than FN1-FGFR1 or FN1-FGF1.</p

DataSheet_2_Case report: Novel NIPBL-BEND2 fusion gene identified in osteoblastoma-like phosphaturic mesenchymal tumor of the fibula.xlsx

Phosphaturic mesenchymal tumor (PMT) is a rare tumor that secretes fibroblast growth factor 23 (FGF23) and causes hypophosphatemia and tumor-induced osteomalacia (TIO). Fusion genes FN1-FGFR1 and FN1-FGF1 have been detected in some PMTs, but the pathogenesis of PMTs without these fusion genes remains unclear. Here, we report a 12-year-old boy with persistent muscle weakness and gait disturbance. Roentgenographic examination revealed a radiolucent lesion with endosteal scalloping in the left fibula, while his serum level of FGF23 was markedly increased. Combined with simple X-ray findings of other body parts, we suspected that TIO was caused by PMT, and resected the tumor. After resection, the serum level of FGF23 started to decrease immediately and normalized within 3 hours after resection, with this being earlier than normalization of the serum phosphorus level. In RNA sequencing, FN1-FGFR1 and FN1-FGF1 were not detected, but a novel NIPBL-BEND2 fusion gene was identified. When we forcedly expressed this fusion gene in HEK293T cells and MG63 cells, cell proliferation was enhanced in both cell lines. Furthermore, Gene set enrichment analysis of HEK293T cells showed significant upregulation of MYC-target genes. Our results suggest that this novel NIPBL-BEND2 fusion gene promotes cell proliferation possibly via the MYC pathway and might be one of the etiologies of PMTs other than FN1-FGFR1 or FN1-FGF1.</p

Kinetic Trapping of Folded Proteins Relative to Aggregates under Physiologically Relevant Conditions

Anfinsen’s thermodynamic hypothesis does not explicitly take into account the possibility of protein aggregation. Here, we introduce a cyclic-perturbation approach to prove that not only the native state but also soluble aggregates of most proteins can be highly populated under mild, physiologically relevant conditions, even at very low concentration. Surprisingly, these aggregates are not necessarily amyloid in nature and are usually not observed in bioactive proteins due to the extremely low kinetic flux from the native state toward a region of the chemical-potential landscape encoding aggregates. We first illustrate this concept for the representative model protein apomyoglobinat room temperature and no denaturantand demonstrate kinetic trapping of the native state relative to at least two different types of soluble, predominantly nonamyloid aggregates. The concentration and temperature dependence of aggregation confirm the above scenario. Extension of our analysis to the Escherichia coli proteome shows that the majority of the soluble bacterial proteome is also kinetically trapped in the nonaggregated state. Hence, the existence and low kinetic accessibility of large aggregates at room temperature and pH 6–7 is a general phenomenon. We also show that the average critical protein concentration for aggregation of most of the bacterial proteome is extremely small, much lower than the typical cellular protein concentration. Hence, the thermodynamic driving force for protein aggregation is large even if aggregation does not usually occur in healthy cells due to kinetic trapping. A broader view of Anfinsen’s thermodynamic hypothesis encompassing all protein states, including aggregates, is necessary to understand the behavior of proteins in their natural environment

Log₂ fold-change of genes with rpkm > 1 in a Tet-BNRF1/Akata(-) cells in the presence or absence of Dox in RNA-seq analyses.

Log2 fold-change of genes with rpkm > 1 in a Tet-BNRF1/Akata(-) cells in the presence or absence of Dox in RNA-seq analyses.</p