Probing the Acid-Induced Packing Structure Changes of the Molten Globule Domains of a Protein near Equilibrium Unfolding

Abstract

Using simultaneously scanning small-angle X-ray scattering (SAXS) and UV-vis absorption with integrated online size exclusion chromatography, supplemental with molecular dynamics simulations, we unveil the long-postulated global structure evolution of a model multidomain protein bovine serum albumin (BSA) during acid-induced unfolding. Our results differentiate three global packing structures of the three molten globule domains of BSA, forming three intermediates I, I, and E along the unfolding pathway. The I-I transition, overlooked in all previous studies, involves mainly coordinated reorientations across interconnected molten globule subdomains, and the transition activates a critical pivot domain opening of the protein for entering into the E form, with an unexpectedly large unfolding free energy change of -9.5 kcal mol, extracted based on the observed packing structural changes. The revealed local packing flexibility and rigidity of the molten globule domains in the E form elucidate how collective motions of the molten globule domains profoundly influence the folding-unfolding pathway of a multidomain protein.

The Genetic Variability of APOE in Different Human Populations and Its Implications for Longevity

“…It is also important to remember that no definitive mechanism for how APOE binds to lipids has been elucidated even though different hypotheses have emerged over the years, especially in relation to the implication of its isoforms in pathological traits. Starting from the concept of “molten globule” [36,51], a hairpin model has been proposed assuming that the protein bends itself so that the LDLR-binding motif is exposed at one extremity of the structure [31,52,53,54,55]. Other studies have suggested a conformational heterogeneity of bound apoE, observing that LDLR binding affinity, while higher in the bound protein than in the lipid-free protein, is modulated by the particle size, its lipid composition, and the presence of other bound lipoproteins [31,52,56,57,58].…”

The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions

“…SAXS data were collected at the 23A SWAXS end-station equipped with an on-line size exclusion-HPLC system (Agilent chromatographic system 1260 series) at the National Synchrotron Radiation Research Center (NSRRC), Taiwan (76). The sample solution (100 l of 10 mg ml Ϫ1 ; corresponding to the low protein concentration instances after HPLC dilution) was injected into the HPLC column with a flow rate of 0.35 ml min Ϫ1 and directed through a quartz capillary (2.0-mm diameter) thermostatted at 303 K for simultaneous SAXS and UVvisible absorption measurements at the same sample position with orthogonal incidences.…”

Exploring the pH-Induced Functional Phase Space of Human Serum Albumin by EPR Spectroscopy

“…A general overview of applicable methods for MG state detection of proteins is given by Kuwajima [22]. A novel promising approach in characterizing acid-unfolded states is the application of small-angle X-ray scattering (SAXS) [23,24]. An intriguing analogy of how protein thermodynamic states can be related to bulk systems was demonstrated by Pande and Rokhsar [25] in a phase diagram where the native protein (N) corresponds to the solid (s), the molten globule (MG) represents the liquid (l) and the unfolded chain (U) represents the vapor state (v) of a protein.…”