Investigating the hydrogen-bonding model of urea denaturation.
The direct binding mechanism for urea-based denaturation of proteins was tested with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM). Thermodynamic measurements of the polymer's hydrophobic collapse were complemented by Fourier transform infrared (FTIR) spectroscopy, Stokes radius measurements, and methylated urea experiments. It was found that the lower critical solution temperature (LCST) of PNIPAM decreased as urea was added to the solution. Therefore, urea actually facilitated the hydrophobic collapse of the macromolecule. Moreover, these thermodynamic measurements were strongly correlated with amide I band data which indicated that the decrease in the LCST was coupled to the direct hydrogen bonding of urea to the amide moieties of the polymer. In addition, the hydrogen bonding was found to be highly cooperative, which is consistent with a cross-linking (bivalent binding) mechanism. Cross-linking was confirmed by Stokes radius measurements below the polymer's LCST using gel filtration chromatography. Finally, phase transition measurements with methylurea, dimethylurea, and tetramethylurea indicated that these substituted compounds caused the LCST of PNIPAM to rise with increasing methyl group content. No evidence could be found for the direct binding of any of these methylated ureas to the polymer amide moieties by FTIR. These results are inconsistent with a direct hydrogen-bonding mechanism for the urea-induced denaturation of proteins.
Sagle
Laura B
LB
Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77843, USA.
Zhang
Yanjie
Y
Litosh
Vladislav A
VA
Chen
Xin
X
Cho
Younhee
Y
Cremer
Paul S
PS
D000178:Acrylamides / Q000032:analysis / Q000737:chemistry*; D000180:Acrylic Resins; D001665:Binding Sites; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011108:Polymers / Q000032:analysis / Q000737:chemistry*; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D044366:Transition Temperature; D014508:Urea / Q000031:analogs & derivatives / Q000737:chemistry*
D000178:Acrylamides; D000180:Acrylic Resins; D009842:Oligopeptides; D011108:Polymers; D011506:Proteins; C052970:poly-N-isopropylacrylamide; C094883:valyl-prolyl-glycyl-valyl-glycine; D014508:Urea