Multistate Equilibrium Unfolding of Escherichia coli Dihydrofolate Reductase: Thermodynamic and Spectroscopic Description of the Native, Intermediate, and Unfolded Ensembles
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- 作者：Roxana M. Ionescu ; Virginia F. Smith ; John C. O'Neill ; Jr. ; and C. Robert Matthews
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：August 8, 2000
- 年：2000
- 卷：39
- 期：31
- 页码：9540 - 9550
- 全文大小：300K
- 年卷期：v.39,no.31(August 8, 2000)
- ISSN：1520-4995
文摘
The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichiacoli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea andtemperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra wererecorded during temperature-induced unfolding at different urea concentrations and during urea-inducedunfolding at different temperatures. The first three vectors obtained by singular-value decomposition ofeach set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model.Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along bothperturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predictsa maximum apparent stability, 
chars/Delta.gif" BORDER=0 >G
NU, of 6 kcal mol-1 at 15 C, an apparent mNU value of 2 kcal mol-1M-1, and an apparent heat capacity change, chars/Delta.gif" BORDER=0 >Cp-NU, of 2.5 kcal mol-1 K-1. The intermediate species hasa maximum stability of approximately 2 kcal mol-1 and a compactness closer to that of the native thanto that of the unfolded state. The population of the intermediate is maximal (~70%) around 50 C andfalls below the limits of detection of 
2 M urea or at temperatures of <35 or >65 C. The fluorescenceproperties of the equilibrium intermediate resemble those of a transient intermediate detected duringrefolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster inthe adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CDspectroscopic properties of the native state reveal the presence of two principal isoforms that differ inligand binding affinities and in the packing of the adenosine-binding domain. The relative populations ofthese species change slightly with temperature and do not depend on the urea concentration, implyingthat the two native isoforms are well-structured and compact. Global analysis of data from multiplespectroscopic probes and several methods of unfolding is a powerful tool for revealing structural andthermodynamic properties of partially and fully folded forms of DHFR.
chars/Delta.gif" BORDER=0 >GNU, of 6 kcal mol-1 at 15 C, an apparent mNU value of 2 kcal mol-1M-1, and an apparent heat capacity change, chars/Delta.gif" BORDER=0 >Cp-NU, of 2.5 kcal mol-1 K-1. The intermediate species hasa maximum stability of approximately 2 kcal mol-1 and a compactness closer to that of the native thanto that of the unfolded state. The population of the intermediate is maximal (~70%) around 50 C andfalls below the limits of detection of 2 M urea or at temperatures of <35 or >65 C. The fluorescenceproperties of the equilibrium intermediate resemble those of a transient intermediate detected duringrefolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster inthe adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CDspectroscopic properties of the native state reveal the presence of two principal isoforms that differ inligand binding affinities and in the packing of the adenosine-binding domain. The relative populations ofthese species change slightly with temperature and do not depend on the urea concentration, implyingthat the two native isoforms are well-structured and compact. Global analysis of data from multiplespectroscopic probes and several methods of unfolding is a powerful tool for revealing structural andthermodynamic properties of partially and fully folded forms of DHFR.