Red
fluorescent proteins with a large Stokes shift offer a limited
autofluorescence background and are used in deep tissue imaging. Here,
by introducing the free amino group in Aequorea victoria, the electrostatic charges of the p-hydroxybenzylidene
imidazolinone chromophore of green fluorescent protein (GFP) have
been altered resulting in an unusual, 85 nm red-shifted fluorescence.
The structural and biophysical analysis suggested that the red shift
is due to positional shift occupancy of Glu222 and Arg96, resulting
in extended conjugation and a relaxed chromophore. Femtosecond transient
absorption spectra exhibited that the excited state relaxation dynamics
of red-shifted GFP (rGFP) (τ4 = 234 ps) are faster
compared to the A. victoria green fluorescent
protein (τ4 = 3.0 ns). The nanosecond time-resolved
emission spectra of rGFP reveal the continuous spectral shift during
emission by a solvent reorientation in the chromophore. Finally, the molecular dynamics simulations
revealed the rearrangement of the hydrogen bond interactions in the
chromophore vicinity, reshaping the symmetric distribution of van
der Waals space to fine tune the GFP structure resulting from highly
red-shifted rGFP.
Helicobacter pylori (H. pylori)a human gastric pathogenforms a major risk factor for the development of various gastric pathologies such as chronic inflammatory gastritis, peptic ulcer, lymphomas of mucosa-associated lymphoid tissues, and gastric carcinoma. The complete eradication of infection is the primary objective of treating any H. pylori-associated gastric condition. However, declining eradication efficiencies, off-target effects, and patient noncompliance to prolong and broad-spectrum antibiotic treatments has spurred the clinical interest to search for alternative effective and safer therapeutic options. As natural compounds are safe and privileged with high levels of antibacterial-activity, previous studies have tested and reported a plethora of such compounds with potential in vitro/in vivo anti-H. pylori activity. However, the mode of action of majority of these natural compounds is unclear. The present study has been envisaged to compile the information of various such natural compounds and to evaluate their binding with histone-like DNAbinding proteins of H. pylori (referred here as Hup) using in silico molecular docking-based virtual screening experiments. Hupbeing a major nucleoid-associated protein expressed by H. pylori plays a strategic role in its survival and persistent colonization under hostile stress conditions. The ligand with highest binding energy with Hupthat is, epigallocatechin-(−)gallate (EGCG)was rationally selected for further computational and experimental testing. The best docking poses of EGCG with Hup were first evaluated for their solution stability using long run molecular dynamics simulations and then using fluorescence and nuclear magnetic resonance titration experiments which demonstrated that the binding of EGCG with Hup is fairly strong (the resultant apparent dissociation constant (k D ) values were equal to 2.61 and 3.29 ± 0.42 μM, respectively).
Real-time live cell imaging and quantification of biothiols dynamics are important for understanding the pathophysiological process. However, it is still challenging in the design and synthesis of rational probes that...
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