Lin-12/Notch Repeat B: The Effects Of Disulfide Bonding And Hydrophobic Residues On Its Autonomous Folding

Abstract

Notch proteins are transmembrane proteins involved in controlling cell differentiation, cell growth, and cell death. An extracellular negative regulatory region (NRR) contains three contiguous LIN-12/Notch Repeats (LNRs) - LNRA, LNRB, and LNRC. Previous work from our lab showed that the total number of residues N-terminal to the first cysteine residue from human Notch1 LNRB (hN1LNRB) was critical for correct disulfide bond formation. Because of its more central location in relation to the rest of the protein, hN1LNRB participates in extensive interactions via its hydrophobic residues in the context of the full Notch protein. When expressed and folded in isolation, some of these residues are expected to be exposed to solvent and possibly contribute to the requirement of the additional N-terminal residues for autonomous in vitro folding with formation of the correct disulfide bonds. To test this hypothesis we mutated W52 to A52 and compared its folding pattern to that of the wild-type hN1LNRB. The effect of the total number of disulfide bonds on the autonomous folding of hN1LNRB was also investigated. In this study, the first of three pairs of disulfide bonds in hN1LNRB was eliminated by mutating C45 and C69 to A. Two mutant forms with two pairs of disulfide bonds were folded in vitro under the same conditions as the wild-type and the folding patterns were compared. The data and comparative analysis we present in this work demonstrate the importance of specific hydrophobic interactions and the total number of disulfide bonds as key determinants for the correct folding of an LNR module in addition to the total number of amino acids and Ca2+ ion coordination within the module.