Probing the urea dependence of residual structure in denatured human α-lactalbumin

Journal article

Backbone ¹⁵N relaxation parameters and ¹⁵N-¹N^N residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA).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. ¹⁵N R₂ values in some regions of all Ala α-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 3₁₀ helices in native α-LA show R₂ values and RDC data consistent with some non-random structural propensities even a 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 α-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: Higman, V; Rösner, H; Ugolini, R; Greene, L; Redfield, C

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Springer
• Journal: Journal of Biomolecular NMR
• Volume: 45
• Issue: 1-2
• Pages: 121-131
• Publication date: 2009-01-01
• Edition: Publisher's version
• DOI: 10.1007/s10858-009-9342-y
• EISSN: 1573-5001
• ISSN: 0925-2738
• Language: English
• Subjects: Biochemistry; Chemistry & allied sciences