scholarly journals Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

2016 ◽  
Vol 18 (8) ◽  
pp. 5819-5831 ◽  
Author(s):  
Zhanglong Liu ◽  
Thomas M. Casey ◽  
Mandy E. Blackburn ◽  
Xi Huang ◽  
Linh Pham ◽  
...  
Keyword(s):  
Spin Labeling ◽  
Conformational Sampling ◽  
Electron Resonance ◽  
Pulsed Epr ◽  
Double Electron ◽  
Conformational Landscape ◽  
Double Electron Electron Resonance ◽  
Hiv 1

The conformational landscape of HIV-1 protease can be characterized by double electron–electron resonance (DEER) spin-labeling.

scholarly journals Spatial domain organization in the HIV-1 reverse transcriptase p66 homodimer precursor probed by double electron-electron resonance EPR

2019 ◽  
Vol 116 (36) ◽  
pp. 17809-17816 ◽  
Author(s):  
Thomas Schmidt ◽  
Charles D. Schwieters ◽  
G. Marius Clore
Keyword(s):  
Reverse Transcriptase ◽  
Spatial Domain ◽  
Rnase H ◽  
Type I ◽  
Electron Resonance ◽  
Domain Organization ◽  
Double Electron ◽  
Double Electron Electron Resonance ◽  
Single Subunit ◽  
Hiv 1

HIV type I (HIV-1) reverse transcriptase (RT) catalyzes the conversion of viral RNA into DNA, initiating the chain of events leading to integration of proviral DNA into the host genome. RT is expressed as a single polypeptide chain within the Gag-Pol polyprotein, and either prior to or following excision by HIV-1 protease forms a 66 kDa chain (p66) homodimer precursor. Further proteolytic attack by HIV-1 protease cleaves the ribonuclease H (RNase H) domain of a single subunit to yield the mature p66/p51 heterodimer. Here, we probe the spatial domain organization within the p66 homodimer using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure a large number of intra- and intersubunit distances between spin labels attached to surface-engineered cysteines. The DEER-derived distances are fully consistent with the structural subunit asymmetry found in the mature p66/p51 heterodimer in which catalytic activity resides in the p66 subunit, while the p51 subunit purely serves as a structural scaffold. Furthermore, the p66 homodimer precursor undergoes a conformational change involving the thumb, palm, and finger domains in one of the subunits (corresponding to the p66 subunit in the mature p66/p51 heterodimer) from a closed to a partially open state upon addition of a nonnucleoside inhibitor. The relative orientation of the domains was modeled by simulated annealing driven by the DEER-derived distances. Finally, the RNase H domain that is cleaved to generate p51 in the mature p66/p51 heterodimer is present in 2 major conformers. One conformer is fully solvent accessible thereby accounting for the observation that only a single subunit of the p66 homodimer precursor is susceptible to HIV-1 protease.

scholarly journals Double Electron-Electron Resonance and 1H-15N HSQC Spectral Analysis as Means to Understand the Effects of Single and Multiple Site Mutations in the HIV-1 protease

2011 ◽  
Vol 100 (3) ◽  
pp. 374a
Author(s):  
Ian Mitchelle ◽  
S. De Vera ◽  
Mandy E. Blackburn ◽  
Luis Galiano ◽  
Adam N. Smith ◽  
...  
Keyword(s):  
Spectral Analysis ◽  
Multiple Site ◽  
Electron Resonance ◽  
Double Electron ◽  
Double Electron Electron Resonance ◽  
Hiv 1

scholarly journals Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

2020 ◽  
Author(s):  
Joe A. Kaczmarski ◽  
Mithun C. Mahawaththa ◽  
Akiva Feintuch ◽  
Ben E. Clifton ◽  
Luke A. Adams ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Binding Site ◽  
Structural Dynamics ◽  
Binding Proteins ◽  
Conformational Sampling ◽  
Evolutionary Trajectory ◽  
Double Electron ◽  
Conformational Landscape ◽  
Protein Change ◽  
Double Electron Electron Resonance

AbstractSeveral enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including double electron-electron resonance (DEER), on reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.

scholarly journals Natural Conformational Sampling of Human TNFα Visualized by Double Electron-Electron Resonance

2017 ◽  
Vol 113 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Bruce Carrington ◽  
William K. Myers ◽  
Peter Horanyi ◽  
Mark Calmiano ◽  
Alastair D.G. Lawson
Keyword(s):  
Conformational Sampling ◽  
Electron Resonance ◽  
Double Electron ◽  
Double Electron Electron Resonance

Characterization of Ionic Clusters in Different Ionically Functionalized Diblock Copolymers by CW EPR and Four-Pulse Double Electron−Electron Resonance

2001 ◽  
Vol 34 (16) ◽  
pp. 5555-5560 ◽  
Author(s):  
M. Pannier ◽  
M. Schöps ◽  
V. Schädler ◽  
U. Wiesner ◽  
G. Jeschke ◽  
...  
Keyword(s):  
Diblock Copolymers ◽  
Ionic Clusters ◽  
Electron Resonance ◽  
Double Electron ◽  
Double Electron Electron Resonance

scholarly journals Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Joe A. Kaczmarski ◽  
Mithun C. Mahawaththa ◽  
Akiva Feintuch ◽  
Ben E. Clifton ◽  
Luke A. Adams ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Dynamics ◽  
Structural Dynamics ◽  
Conformational Sampling ◽  
Evolutionary Trajectory ◽  
Body Protein ◽  
Double Electron ◽  
Conformational Landscape ◽  
Protein Change ◽  
Double Electron Electron Resonance

AbstractSeveral enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron–electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.

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