Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.
Two of the most common forms of chemical
modifications that compromise
the efficacy of therapeutic proteins are the deamidation of asparagine
residues and oxidation of methionine residues. We probed how deamidation
affects the structure, stability, aggregation, and function of interferon
alpha-2a (IFNA2a), and compared with our earlier results on methionine
oxidation. Upon deamidation, no significant changes were observed
in the global secondary structure of IFNA2a with minor changes in
its tertiary structure. However, deamidation destabilized the protein,
and increased its propensity to aggregate under accelerated stress
conditions. Cytopathic inhibition and antiproliferation assays showed
drastic decrease in the functionality of deamidated IFNA2a compared
to the wild-type. 2D NMR measurements showed structural changes in
local protein regions, with no effect on the overall global structure
of IFNA2a. These local protein regions corresponded well with the
aggregation hot-spots predicted by computational programs, and the
functional hot-spots identified by site-directed mutagenesis. When
compared to the effects of methionine oxidation, deamidation caused
lesser aggregation, because of lesser structural unfolding observed
in aggregation hot-spots by 2D NMR. In comparison to oxidation, deamidation
showed larger decrease in function, because deamidation affected key
amino acid residues in functional hot-spots as observed by 2D NMR
and structural modeling. Such quantitative comparison between the
effects of deamidation and oxidation on a pharmaceutical protein has
not been done before, and the high-resolution structural information
on local protein regions obtained by 2D NMR provided a better insight
compared to low-resolution methods that probe global protein structure.
Binding immunoglobulin protein (BiP) is a molecular chaperone important for the folding of numerous proteins, which include millions of immunoglobulins in human body. It also plays a key role in the unfolded protein response (UPR) in the endoplasmic reticulum. Free radical generation is a common phenomenon that occurs in cells under healthy as well as under stress conditions such as ageing, inflammation, alcohol consumption, and smoking. These free radicals attack the cell membranes and generate highly reactive lipid peroxidation products such as 4-oxononenal (4-ONE). BiP is a key protein that is modified by 4-ONE. In this study, we probed how such chemical modification affects the biophysical properties of BiP. Upon modification, BiP shows significant tertiary structural changes with no changes in its secondary structure. The protein loses its thermodynamic stability, particularly, that of the nucleotide binding domain (NBD) where ATP binds. In terms of function, the modified BiP completely loses its ATPase activity with decreased ATP binding affinity. However, modified BiP retains its immunoglobulin binding function and its chaperone activity of suppressing non-specific protein aggregation. These results indicate that 4-ONE modification can significantly affect the structure-function of key proteins such as BiP involved in cellular pathways, and provide a molecular basis for how chemical modifications can result in the failure of quality control mechanisms inside the cell.
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