More Like this

1. An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET

   https://doi.org/10.7554/eLife.70236  

   Zagotta, William N ; Sim, Brandon S ; Nhim, Anthony K ; Raza, Marium M ; Evans, Eric GB ; Venkatesh, Yarra ; Jones, Chloe M ; Mehl, Ryan A ; Petersson, E James ; Gordon, Sharona E ( October 2021 , eLife)

   With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuringmore »the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.« less
2. Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy

   https://doi.org/10.3390/ijms20215308  

   Bonanno, Alexander ; Blake, Robert C. ; Chong, Parkson Lee-Gau ( November 2019 , International Journal of Molecular Sciences)

   In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVsmore »reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.« less
3. Determining the concentration and enantiomeric composition of histidine using one fluorescent probe

   https://doi.org/10.1039/d0cc07498e  

   Mao, Yifan ; Abed, Mehdi A. ; Lee, Nathan B. ; Wu, Xuedan ; Du, Gengyu ; Pu, Lin ( January 2021 , Chemical Communications)

   A chemoselective as well as enantioselective fluorescent probe has been developed to determine both the concentration and enantiomeric composition of the biologically important amino acid histidine by measuring the fluorescence responses when excited at two different wavelengths.
4. Vinculin tension probe in neurons

   https://doi.org/10.1117/12.2578501  

   Ayad, Marina A. ; Mahon, Timothy ; Sumetsky, Daniel ; Cararo-Lopes, Marina M. ; Firestein, Bonnie L. ; Boustany, Nada N. ( March 2021 , Optical Techniques in Neurosurgery, Neurophotonics, and Optogenetics)

   Yang, Victor X. ; Kainerstorfer, Jana M. ; Luo, Qingming ; Ding, Jun ; Fu, Ling ; Mohanty, Samarendra K. ; Roe, Anna W. ; Shoham, Shy (Ed.)

   Vinculin is a known key regulator of focal adhesions; it undergoes tension in the locations of attachment to the extracellular matrix. In this study, we explore the use of a vinculin tension FRET probe to investigate vinculin tension within neurons. A critical component of neuronal growth is migration, which is dependent on the mechanical cues between the cells and the extracellular matrix. An understanding of tension variation within the neuron may help us understand mechanisms of neurogenesis. To study these forces, we use a previously developed molecular tension sensor, which consists of an elastic linker, TSMod, a 40-amino-acid-long peptide inserted between teal fluorescent protein (mTFP1) and mVenus. The vinculin tension sensor, VinTS, consists of TSMod embedded between the Vinculin head and tail. When under tension, VinTS will exhibit a lower fluorescence resonance energy transfer (FRET) efficiency between mTFP1 and mVenus. Cortical neurons were isolated from embryonic rat brains and cultured on glass coverslips coated with poly-D-lysine and laminin. The neurons were transfected with TSMod (the unloaded tension sensor) or VinTS. Neurons expressing TSMod are used as the experiment’s control group since TSMod on its own is not affected by vinculin tension. The mean FRET efficiency of 171 TSMod and 127more »VinTS expressing neurons was 27.08 ± 4.98%, and 22.86 ± 3.98%, respectively. The FRET efficiency of VinTS was significantly lower than that of TSMod (p = 6.6e15 by Welch’s t-test). These results support the feasibility of using the VinTS probe in neurons and provide a first assessment of VinTS FRET efficiency in neurons. The lower FRET efficiency of VinTS compared with TSMod suggests that VinTS may be under tension in neurons. However, additional studies are required to further characterize these results.« less
5. Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP

   https://doi.org/10.3390/ijms20205229  

   Sen, Tirthendu ; Mamontova, Anastasia ; Titelmayer, Anastasia ; Shakhov, Aleksander ; Astafiev, Artyom ; Acharya, Atanu ; Lukyanov, Konstantin ; Krylov, Anna ; Bogdanov, Alexey ( October 2019 , International Journal of Molecular Sciences)

   Enhanced green fluorescent protein (EGFP)—one of the most widely applied genetically encoded fluorescent probes—carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion (“redding”) with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss ofmore »quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.« less