A Dynamical Investigation of Acrylodan-Labeled Mutant Phosphate Binding Protein

Abstract

The static and dynamical behavior of a fluorescently labeled mutant of the Escherichia coli periplasmic phosphate binding protein (PBP) was investigated through steady-state and time-resolved fluorescence spectroscopy. As a means of developing a biorecognition element for inorganic phosphate (P(i)), alanine-197 of PBP was replaced with a cysteine. This site was then labeled with an environmentally sensitive fluorophore. The fluorescence emission of the mutant PBP labeled with acrylodan (MPBP-AC) proved to be sensitive to micromolar concentrations of P(i), as indicated by a 50% increase in the steady-state emission intensity. Steady-state results indicated that the labeling protocol was specific for cys-197 only and did not label the wild-type PBP; thus, a site-selective labeling protocol was developed. Time-resolved measurements were used to determine the influence of the dynamics of MPBP-AC on the process of signal transduction. Time-resolved anisotropy measurements revealed that rotational dynamics were best described by a model with two independent motions: the global motion of the protein and the local motion of the acrylodan probe. The rates of both global and local rotational reorientation of MPBP-AC were faster when the protein was P(i)-bound rather than P(i)-free. This was a result of structural changes involving or surrounding both the P(i)-binding site (global changes) and the residues in near proximity to the fluorescent reporter group (local changes). Recovery of the semiangle (theta) indicated that local structural changes in MPBP-AC took place when P(i) was bound to the protein. Acrylodan gained mobility when MPBP-AC bound P(i), as indicated by the fact that theta increased by approximately 5 degrees. In addition, dynamic quenching measurements confirmed that structural changes occurred locally near the cys-197. Acrylodan became more accessible to iodide when MPBP-AC bound P(i), as demonstrated by the 35% increase in the value of the bimolecular quenching constant.

A Novel Reagentless Sensing System for Measuring Glucose Based on the Galactose/Glucose-Binding Protein

“…Similar work performed in our laboratory in the rational design of a sensing system for calcium also pointed to the same reason regarding the behavior of the probes (21,22). In addition, we have also studied the structure and dynamics of a mutant form of the phosphate-binding protein that was site specifically labeled with MDCC (23). We conclude that the quenching of the fluorescence signal seen in this study is a result of the probe being more exposed to the solvent or a hydrophilic environment when the protein binds glucose.…”

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli

“…Studies of intrinsic tryptophan quenching in NBP upon binding to a ligand also demonstrated a strong response to cobalt [22]. The remaining metals all showed less maximum quenching than nickel and detection limits were more than 10 to 100 times higher than for nickel.…”

Optical Determination of Glutamine Using a Genetically Engineered Protein

“…A number of E. coli periplasmic binding proteins have been utilized for the development of potential biosensors for phosphate (22)(23)(24), glucose (25,26), and maltose (27). Due to the favorable sensitivity and specificity for glutamine, GlnBP was selected as the basis for the optical biosensor.…”