Protein Engineering, Vol. 12, No. 7, 581-587,
July 1999
© 1999 Oxford University Press
Stability, activity and flexibility in 
-lactalbumin
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida, FL 33101, USA and 2 Department of Biology and Biochemistry, University of Bath, Bath, UK
-Lactalbumins and the type-c lysozymes are homologues with similar folds that differ in function and stability. To determine if the lower stability of -lactalbumin results from specific substitutions required for its adaptation to a new function, the effects of lysozyme-based and other substitutions on thermal stability were determined. Unblocking the upper cleft in -lactalbumin by replacing Tyr103 with Ala, perturbs stability and structure but Pro, which also generates an open cleft, is compatible with normal structure and activity. These effects appear to reflect alternative enthalpic and entropic forms of structural stabilization by Tyr and Pro. Of 23 mutations, only three, which involve substitutions for residues in flexible substructures adjacent to the functional site, increase stability. Two are lysozyme-based substitutions for Leu110, a component of a region with alternative helix and loop conformations, and one is Asn for Lys114, a residue whose microenvironment changes when -lactalbumin interacts with its target enzyme. While all substitutions for Leu110 perturb activity, a Lys114 to Asn mutation increases Tm by more than 10°C and reduces activity, but two other destabilizing substitutions do not affect activity. It is proposed that increased stability and reduced activity in Lys114Asn result from reduced flexibility in the functional site of -lactalbumin.
Keywords: flexibility/-lactalbumin/lactose synthase/lysozyme/protein structure/thermal stability
3 To whom correspondence should be addressed
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