The thermostability of an alpha-helical coiled-coil protein and its potential use in sensor applications.
Coiled-coil proteins are assemblies of two to four alpha-helices that pack together in a parallel or anti-parallel fashion. Coiled-coil structures can confer a variety of functional capabilities, which include enabling proteins, such as myosin, to function in the contractile apparatus of muscle and non-muscle cells. The TlpA protein encoded by the virulence plasmid of Salmonella is an alpha-helical protein that forms an elongated coiled-coil homodimer. A number of studies have clearly established the role of TlpA as a temperature-sensing gene regulator, however the potential use of a TlpA in a thermo-sensor application outside of the organism has not been exploited. In this paper, we demonstrate that TlpA has several characteristics that are common with alpha-helical coiled-coils and its thermal folding and unfolding is reversible and rapid. TlpA is extremely sensitive to changes in temperature. We have also compared the heat-stability of TlpA with other structurally similar proteins. Using a folding reporter, in which TlpA is expressed as a C-terminal fusion with green fluorescent protein (GFP), we were able to use fluorescence as an indicator of folding and unfolding of the fusion protein. Our results on the rapid conformational changes inherent in TlpA support the previous findings and we present here preliminary data on the use of a GFP-TlpA fusion protein as temperature sensor.
Naik
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RR
Air Force Research Laboratory, Materials and Manufacturing Directorate, Wright-Patterson Air Force Base, OH 45433-7702, USA.
Kirkpatrick
S M
SM
Stone
M O
MO
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics; D013816:Thermodynamics
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C075138:TlpA protein, Salmonella