A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds

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Title: A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds
Author: Evans, Matthew Ryan
Abstract: The aryl hydrocarbon receptor nuclear translocator (ARNT ) is a promiscuous basic helix -loop -helix Period /ARNT /Single -minded (bHLH -PAS ) protein that controls various biological pathways by forming heterodimeric transcriptional regulator complexes with several other bHLH -PAS proteins via the beta -sheet surfaces of its two PAS domains . The beta -sheets of PAS domains are involved in many intermolecular interactions with other proteins and natural cofactors in order to detect environmental changes in sensor PAS proteins . As part of a study of the HIF -2 alpha and ARNT PAS -B heterodimer , site -directed mutagenesis was performed on the ARNT PAS -B domain . Interestingly , one point mutation on the ARNT beta -sheet surface (Y456T ) resulted in a new conformation of the domain that existed in equimolar concentrations with the wild -type conformation . Subsequent studies demonstrated that the two conformations are in equilibrium and that relative populations of the two conformations can be perturbed by additional mutations . Using solution NMR spectroscopy , we solved the high resolution solution structure of a mutant ARNT PAS -B domain in the new conformation , demonstrating that it contains a three -residue slip in register and accompanying inversion of the central beta -strand . In addition , this new conformer has a greater than hundred -fold reduction in affinity for HIF -2 alpha PAS -B , disrupting the hypoxia response pathway . Solution NMR measurements of the interconversion kinetics have let us establish that these two conformations interconvert slowly (40 min at RT ) with a linear Arrhenius temperature -dependence of the interconversion rate . Stopped -flow unfolding experiments using GdmHCl on Y456T , revealed a similarly slow unfolding rate (25 min at RT ) and an energy barrier to unfold of approximately 13 kcal /mol , which is nearly identical to that for the interconversion process . These data indicate that the protein must undergo a global unfolding process in order to interconvert between conformations . Lastly , these relative populations of Y456T can be affected by compound preferentially binding into the core of one of these conformations . This discovery highlights the malleability of PAS beta -sheets and suggests ARNT may act as a regulatory switch to control different biological pathways . Furthermore , this system presents a great opportunity to further understand the structural and kinetic impact of beta -strand slips observed in nature .
URI: http : / /hdl .handle .net /2152 .5 /749
Date: 2009-09-04

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A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /749 .

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