The Effects of Reduction Potential and Number of Disulfide Bonds on the Correct Folding of Lin-12/Notch Repeats (LNRs) Using Human Notch 1 LNRA as a Model System

Abstract

Notch receptors are multi-domain trans-membrane proteins that are important for cell-cell communication and development. Deregulated Notch signaling has been linked to many human diseases such as sclerosis, artereopathy and leukemia. The extra-cellular domain of the Notch Receptor contains the Ligand Binding Domain and the Negative Regulatory Region (NRR), which includes three Lin-12/Notch Repeats (LNR), small disulfide-rich sequences of 35 residues. It has been previously shown that the first LNR from human Notch1, hN1LNRA, requires Ca2+ and a certain reduction potential that ensures the correct formation of three specific disulfide bonds believed to be critical for LNR structure and function. However, the first LNR in human Notch 4 and some of the LNRs found in PAPP (pregnancy-associated plasma protein-A), only possess four cysteines thereby can only form two disulfide bonds.In this work we present our findings on the effect of various reduction potentials as well as the elimination of the first disulfide bond in the in vitro folding of hN1 LNRA through a comparative analysis. The kinetics of the folding process for both the wild-type and the four-cysteine mutant form of hN1LNRA is studied by trapping various folding intermediates in a time-course manner, which is possible due to the slow rate of disulfide bond formation. Our results indicate that even though the wild-type hN1LNRA is very tolerant to variations in the specific redox potential in obtaining its ultimate correct folding, the its folding kinetics is significantly impacted. This is in contrast to the mutant form, which does not fold into a single species under identical refolding conditions.