Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease

Sternisha, Shawn M.; Whittington, A. Carl; Fiesco, Juliana A. Martinez; Porter, Carol M.; McCray, Malcolm M.; Logan, Timothy M.; Olivieri, Cristina; Veglia, Gianluigi; Steinbach, Peter; Miller, Brian G.

doi:10.1016/j.bpj.2019.12.036

Cited by 12 publications

(7 citation statements)

References 79 publications

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“…Data fitting to determine fluorescence lifetimes was performed using the maximum entropy method as implemented in the MemExp software (ver. 6.0). , We calculated amplitude average fluorescence lifetimes ⟨τ⟩ and the radiative and nonradiative rate constants for relaxation, k f and k nr , respectively …”

Section: Resultsmentioning

confidence: 99%

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Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue ^DEAtC

Turner,

Cizmic,

Rosansky

et al. 2023

Bioconjugate Chem.

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Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to livecell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEA tC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson−Crick base pairing. The DEA tCcontaining DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEA tC base paired with G and little fluorescence turn-on when DEA tC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern−Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA−RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEA tC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

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Section: Resultsmentioning

confidence: 99%

“…6.0). 48,49 We calculated amplitude average fluorescence lifetimes ⟨τ⟩ and the radiative and nonradiative rate constants for relaxation, k f and k nr , respectively. 39 In most observed contexts, the DEA tC nucleoside's excited state exhibits biexponential decay.…”

Section: ■ Introductionmentioning

confidence: 99%

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue ^DEAtC

Turner,

Cizmic,

Rosansky

et al. 2023

Bioconjugate Chem.

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“…Correlation functions were analyzed in terms of distributed diffusion times τ D using the program MemDif. Written for this work, MemDif was adapted from MemExp [ 73 ], which has proven very useful in recovering distributions of lifetimes from noisy kinetics data [ 74 , 75 , 76 ]. MemDif and MemExp are available online.…”

Section: Methodsmentioning

confidence: 99%

Concanavalin A Delivers a Photoactive Protein to the Bacterial Wall

Mussini,

Delcanale,

Berni

et al. 2024

IJMS

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Modular supramolecular complexes, where different proteins are assembled to gather targeting capability and photofunctional properties within the same structures, are of special interest for bacterial photodynamic inactivation, given their inherent biocompatibility and flexibility. We have recently proposed one such structure, exploiting the tetrameric bacterial protein streptavidin as the main building block, to target S. aureus protein A. To expand the palette of targets, we have linked biotinylated Concanavalin A, a sugar-binding protein, to a methylene blue-labelled streptavidin. By applying a combination of spectroscopy and microscopy, we demonstrate the binding of Concanavalin A to the walls of Gram-positive S. aureus and Gram-negative E. coli. Photoinactivation is observed for both bacterial strains in the low micromolar range, although the moderate affinity for the molecular targets and the low singlet oxygen yields limit the overall efficiency. Finally, we apply a maximum entropy method to the analysis of autocorrelation traces, which proves particularly useful when interpreting signals measured for diffusing systems heterogeneous in size, such as fluorescent species bound to bacteria.

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“…Between the two domains is a cleft forming the active site where glucose binds. The orientation of the two domains is not static, as GCK exists in multiple conformational ensembles [ 18 , 19 ]. These ensembles are often described as three conformations: the super-open, open and closed conformation [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

A comprehensive map of human glucokinase variant activity

et al. 2023

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Background Glucokinase (GCK) regulates insulin secretion to maintain appropriate blood glucose levels. Sequence variants can alter GCK activity to cause hyperinsulinemic hypoglycemia or hyperglycemia associated with GCK-maturity-onset diabetes of the young (GCK-MODY), collectively affecting up to 10 million people worldwide. Patients with GCK-MODY are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. Result Here, we exploit a multiplexed yeast complementation assay to measure both hyper- and hypoactive GCK variation, capturing 97% of all possible missense and nonsense variants. Activity scores correlate with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and with evolutionary conservation. Hypoactive variants are concentrated at buried positions, near the active site, and at a region of known importance for GCK conformational dynamics. Some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. Conclusion Our comprehensive assessment of GCK variant activity promises to facilitate variant interpretation and diagnosis, expand our mechanistic understanding of hyperactive variants, and inform development of therapeutics targeting GCK.

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Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease

Cited by 12 publications

References 79 publications

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue ^DEAtC

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue ^DEAtC

Concanavalin A Delivers a Photoactive Protein to the Bacterial Wall

A comprehensive map of human glucokinase variant activity

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Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease

Cited by 12 publications

References 79 publications

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC

Concanavalin A Delivers a Photoactive Protein to the Bacterial Wall

A comprehensive map of human glucokinase variant activity

Contact Info

Product

Resources

About

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue ^DEAtC

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue ^DEAtC