Solution Structure and Dynamics of a Heat Shock Protein Assembly Probed by Hydrogen Exchange and Mass Spectrometry
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- 作者：Patrick L. Wintrode ; Kenneth L. Friedrich ; Elizabeth Vierling ; Jean B. Smith ; and David L. Smith
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：September 16, 2003
- 年：2003
- 卷：42
- 期：36
- 页码：10667 - 10673
- 全文大小：296K
- 年卷期：v.42,no.36(September 16, 2003)
- ISSN：1520-4995
文摘
The solution conformation and dynamics of the 16.9 kDa small heat shock protein from wheathave been studied using a combination of hydrogen/deuterium exchange, proteolytic digestion, and massspectrometry. At room temperature, HSP16.9 exists as a dodecameric assembly. Regions of HSP16.9 thatform extensive and essential intersubunit contacts in the assembly, including residues 1-40 and 131-151, show little or no protection against hydrogen/deuterium exchange after incubation in D2O for 5 s.The high levels of hydrogen/deuterium exchange indicate that these regions have experienced largeconformational fluctuations in solution, breaking intersubunit contacts and exposing buried amide hydrogensto solvent. When HSP16.9 is pulse labeled for 10 ms, residues 1-40 and 131-151 are substantially moreprotected than they are after 5 s. Thus, the breaking of intersubunit contacts occurs on a time scale between10 milliseconds and 5 s. At 42 
C, HSP16.9 exists in a suboligomeric form. When the intrinsic temperaturedependence of hydrogen/deuterium exchange is taken into account, exchange patterns at 25 and 42 Care identical within experimental error, suggesting that the conformation of individual HSP16.9 subunitsis the same in both the dodecameric and subdodecameric forms. Significant protection is seen in regionsthat form the dimeric interface, suggesting that the stable suboligomeric form is a dimer. Taken together,these results suggest that heat activation of HSP16.9 occurs by shifting the dodecamer 
dimer equilibriumin favor of free dimers. The conformation of the dimers themselves does not appear to be altered with anincrease in temperature.
C, HSP16.9 exists in a suboligomeric form. When the intrinsic temperaturedependence of hydrogen/deuterium exchange is taken into account, exchange patterns at 25 and 42 Care identical within experimental error, suggesting that the conformation of individual HSP16.9 subunitsis the same in both the dodecameric and subdodecameric forms. Significant protection is seen in regionsthat form the dimeric interface, suggesting that the stable suboligomeric form is a dimer. Taken together,these results suggest that heat activation of HSP16.9 occurs by shifting the dodecamer dimer equilibriumin favor of free dimers. The conformation of the dimers themselves does not appear to be altered with anincrease in temperature.