Isothermal Chemical Denaturation Research Articles

Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide‐binding domain 1

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T(m)) by 6-7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) <==> I(T)(±MgATP) → A(T) → (A(T))(n). The equilibrium unfolding to intermediate, I(T), is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A(T), for which aggregation to (A(T))(n) and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A(T).

Iterative Protein Redesign

An iterative redesign protocol for the transformation of a non-native peptide into a series of nativelike proteins derived from elementary considerations of biological evolution coupled with 1H NMR as an artificial selection criterion is presented. Each of three heptad d position leucines in the helix−helix interfaces of the prototype heme protein maquette, [H10H24]2 or (α-SS-α)2, were replaced in a unit modification per helix by more conformationally restricted β-branched and aromatic amino acids. The secondary structure content (evaluated by circular dichroism and infrared spectroscopies), solvent accessibility of the tryptophan residues (measured by fluorescence spectroscopy), global stability (quantitated by isothermal chemical denaturation), and degree of conformational specificity (determined by 1H NMR spectroscopy) of the resultant peptides were determined. Improvement in the degree of conformational specificity was utilized as a selection criterion to choose three of the nine singly modified pepti...