title: Probing the urea dependence of residual structure in denatured human alpha-lactalbumin.
abstract: Backbone (15)N relaxation parameters and (15)N-(1)H(N) residual dipolar couplings (RDCs) have been measured for a variant of human alpha-lactalbumin (alpha-LA) in 4, 6, 8 and 10 M urea. In the alpha-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala alpha-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. (15)N R(2) values in some regions of all-Ala alpha-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 3(10) helices in native alpha-LA show R(2) values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101-106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala alpha-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant.
authors.affiliation: Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, UK.
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Higman
authors.forename: Heike I
authors.initials: HI
authors.lastname: Rösner
authors.forename: Raffaella
authors.initials: R
authors.lastname: Ugolini
authors.forename: Lesley H
authors.initials: LH
authors.lastname: Greene
authors.forename: Christina
authors.initials: C
authors.lastname: Redfield
authors.forename: Lorna J
authors.initials: LJ
authors.lastname: Smith
mesh_terms: D000409:Alanine / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011489:Protein Denaturation; D017510:Protein Folding; D014508:Urea / Q000737:chemistry*
chemical_list: D009587:Nitrogen Isotopes; D014508:Urea; D007768:Lactalbumin; D000409:Alanine