Backbone Dynamics of the N-Terminal Domain in E. Coli DnaJ Determined by 15N- and 13CO-Relaxation Measurements
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- 作者：Kai Huang ; Ranajeet Ghose ; John M. Flanagan ; and James H. Prestegard
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 10, 1999
- 年：1999
- 卷：38
- 期：32
- 页码：10567 - 10577
- 全文大小：176K
- 年卷期：v.38,no.32(August 10, 1999)
- ISSN：1520-4995
文摘
The backbone dynamics of the N-terminal domain of the chaperone protein Escherichia coliDnaJ have been investigated using steady-state 1H-15N NOEs, 15N T1, T2, and T1
relaxation times, steady-state 13C
-13CO NOEs, and 13CO T1 relaxation times. Two recombinant constructs of the N-terminaldomain of DnaJ have been studied. One, DnaJ(1-78), contains the most conserved "J-domain" of DnaJ,and the other, DnaJ(1-104), includes a glycine/phenylalanine rich region ("G/F" region) in addition tothe "J-domain". DnaJ(1-78) is not capable of stimulating ATP hydrolysis by DnaK, despite the fact thatall currently identified sites responsible for DnaJ-DnaK interaction are located in this region. DnaJ(1-104), on the other hand, retains nearly the full ATPase stimulatory activity of full length DnaJ. Recently,a structural analysis of these two molecules was presented in an effort to elucidate the origin of theirfunctional differences [Huang, K., Flanagan, J. M., and Prestegard, J. H. (1999) Protein Science 8, 203-214]. Herein, an analysis of dynamic properties is presented in a similar effort. A generalized model-freeapproach with a full treatment of the anisotropic overall rotation of the proteins is used in the analysis ofmeasured relaxation parameters. Our results show that internal motions on pico- to nanosecond time scalesin the backbone of DnaJ(1-78) are reduced on the inclusion of the "G/F" region, while conformationalexchange on micro- to millisecond time scales increases. We speculate that the enhanced flexibility ofresidues on the slow time scale upon the inclusion of the "G/F" region could be relevant to the ATPasestimulatory activity of DnaJ if an "induced-fit" mechanism applies to DnaJ-DnaK interactions.
relaxation times, steady-state 13C-13CO NOEs, and 13CO T1 relaxation times. Two recombinant constructs of the N-terminaldomain of DnaJ have been studied. One, DnaJ(1-78), contains the most conserved "J-domain" of DnaJ,and the other, DnaJ(1-104), includes a glycine/phenylalanine rich region ("G/F" region) in addition tothe "J-domain". DnaJ(1-78) is not capable of stimulating ATP hydrolysis by DnaK, despite the fact thatall currently identified sites responsible for DnaJ-DnaK interaction are located in this region. DnaJ(1-104), on the other hand, retains nearly the full ATPase stimulatory activity of full length DnaJ. Recently,a structural analysis of these two molecules was presented in an effort to elucidate the origin of theirfunctional differences [Huang, K., Flanagan, J. M., and Prestegard, J. H. (1999) Protein Science 8, 203-214]. Herein, an analysis of dynamic properties is presented in a similar effort. A generalized model-freeapproach with a full treatment of the anisotropic overall rotation of the proteins is used in the analysis ofmeasured relaxation parameters. Our results show that internal motions on pico- to nanosecond time scalesin the backbone of DnaJ(1-78) are reduced on the inclusion of the "G/F" region, while conformationalexchange on micro- to millisecond time scales increases. We speculate that the enhanced flexibility ofresidues on the slow time scale upon the inclusion of the "G/F" region could be relevant to the ATPasestimulatory activity of DnaJ if an "induced-fit" mechanism applies to DnaJ-DnaK interactions.