Our aim is to determine whether the disulfide bonds of alpha-lactalbumin account for the lack of cooperative folding behavior reported for some molten globule variants, in contrast to the highly cooperative folding reported for the pH 4 molten globule of apomyoglobin. Two different alpha-lactalbumin genetic constructs are studied: [28-111], which has a single disulfide bond connecting two segments of the alpha-helix domain, and [all-Ala], which has no disulfide bonds. The superposition test used earlier to probe for cooperative folding of the apomyoglobin molten globule is used to determine whether there is an important difference in folding cooperativity between the molten globules of [28-111] and [all-Ala]. The [all-Ala] construct behaves in the same manner as the apomyoglobin molten globule: its folding satisfies the superposition test in the three sets of anion conditions studied, and anions stabilize it against urea unfolding. The [28-111] construct behaves differently in both respects: the folding of its molten globule does not satisfy the superposition test in two of the three sets of anion conditions, and anions barely affect its stability. The 28-111 disulfide bond stabilizes the molten globule substantially, as expected from earlier work. Comparison of the unfolding transition curves monitored by circular dichroism also demonstrates that [28-111] folds in a less cooperative manner than [all-Ala]: the unfolding curve of [28-111] is significantly broader. Moreover, the unfolding curves indicate that [28-111] has a lower helix content than [all-Ala]. Consequently, the 28-111 bond constrains the folding behavior of the molten globule and weakens its cooperativity of folding. Study holds ProTherm entries: 13168, 13169, 13170, 13171, 13172, 13173 Extra Details: Alpha-lactalbumin(all-Ala) disulfide bonds; alpha-lactalbumin; molten globule variants;,urea unfolding; circular dichroism
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||12|
|Proteins||Alpha-lactalbumin ; Alpha-lactalbumin|
|Assays/Quantities/Protocols||Experimental Assay: Cm ionic:NaClO4: 50 mM ; Experimental Assay: m ionic:NaClO4: 50 mM ; Experimental Assay: Cm ionic:Na2SO4: 20 mM ; Experimental Assay: m ionic:Na2SO4: 20 mM ; Experimental Assay: Cm ionic:NaCl: 30 mM ; Experimental Assay: m ionic:NaCl: 30 mM|
|Libraries||Mutations for sequence MRFFVPLFLVGILFPAILAKQFTKCELSQLLKDIDGYGGIALPELICTMFHTSGYDTQAIVENNESTEYGLFQISNKLWCKSSQVPQSRNICDISCDKFLDDDITDDIMCAKKILDIKGIDYWLAHKALCTEKLEQWLCEKL|