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

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 <b>I</b>_<b>1</b>, <b>I</b>_<b>2</b>, and <b>E</b> along the unfolding pathway. The <b>I</b>_<b>1</b>–<b>I</b>_<b>2</b> 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 <b>E</b> form, with an unexpectedly large unfolding free energy change of −9.5 kcal mol^–1, extracted based on the observed packing structural changes. The revealed local packing flexibility and rigidity of the molten globule domains in the <b>E</b> form elucidate how collective motions of the molten globule domains profoundly influence the folding–unfolding pathway of a multidomain protein