title: Distinguishing specific and nonspecific interdomain interactions in multidomain proteins.
abstract: Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices. Several of these domains have been shown to be stabilized by their neighbors. Until now, this stabilization has been attributed to specific interactions between the natural neighbors, however we have recently observed that nonnatural neighboring domains can also induce a significant amount of stabilization. Here we investigate this nonnative stabilizing effect. We created spectrin-titin domain pairs of both spectrin R16 and R17 with a single titin I27 domain at either the N- or the C-terminus and found that spectrin domains are significantly stabilized, through slowed unfolding, by nonnative interactions at the C-terminus only. Of particular importance, we show that specific interactions between natural folded neighbors at either terminus confer even greater stability by additionally increasing the folding rate constants. We demonstrate that it is possible to distinguish between natural stabilizing interactions and nonspecific stabilizing effects through examination of the kinetics of well chosen mutant proteins. This work adds to the complexity of studying multidomain proteins.
authors: Cambridge University Chemical Laboratory, MRC Centre for Protein Engineering, Cambridge, United Kingdom.
authors: Lucy G
authors: LG
authors: Randles
authors: Sarah
authors: S
authors: Batey
authors: Annette
authors: A
authors: Steward
authors: Jane
authors: J
authors: Clarke
mesh_terms: D000818:Animals; D002645:Chickens; D064211:Connectin; D006801:Humans; D007700:Kinetics; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013049:Spectrin / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D013049:Spectrin; D011494:Protein Kinases