The structural basis of urea-induced protein unfolding in β-catenin.
Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of β-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic interactions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation.
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Chao
C
Wang
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Zhongzhou
Z
Chen
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Xia
X
Hong
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Fangkun
F
Ning
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Haolin
H
Liu
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Jianye
J
Zang
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Xiaoxue
X
Yan
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Jennifer
J
Kemp
Department of Pharmacology, Anschutz Medical Campus, University of Colorado, Aurora, CO 80045, USA.
Catherine A
CA
Musselman
Department of Pharmacology, Anschutz Medical Campus, University of Colorado, Aurora, CO 80045, USA.
Tatinna G
TG
Kutateladze
Department of Biochemistry and Molecular Genetics, Anschutz Medical Campus, University of Colorado, Aurora, CO 80045, USA.
Rui
R
Zhao
Chinese Academy of Medical Sciences, Department of Biochemistry and Molecular Biology, Peking Union Medical College, Tsinghua University, Beijing 100005, People's Republic of China.
Chengyu
C
Jiang
The Integrated Department of Immunology, National Jewish Health, Denver, CO 80206, USA.
Gongyi
G
Zhang
D000818:Animals; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D051379:Mice; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D058767:Protein Unfolding; D014508:Urea / Q000737:chemistry*; D014867:Water / Q000737:chemistry; D051176:beta Catenin / Q000737:chemistry*
D051176:beta Catenin; D014867:Water; D014508:Urea
hydrogen bonds; urea; β-catenin