Homology Modeling and Molecular Dynamics Simulation Combined with X‑ray Solution Scattering Defining Protein Structures of Thromboxane and Prostacyclin Synthases

A combination of molecular dynamics (MD) simulations and X-ray scattering (SAXS) has emerged as the approach of choice for studying protein structures and dynamics in solution. This approach has potential applications for membrane proteins that neither are soluble nor form crystals easily. We explore the water-coupled dynamic structures of thromboxane synthase (TXAS) and prostacyclin synthase (PGIS) from scanning HPLC–SAXS measurements combined with MD ensemble analyses. Both proteins are heme-containing enzymes in the cytochrome P450 family, known as prostaglandin H₂ (PGH₂) isomerase, with counter-functions in regulation of platelet aggregation. Currently, the X-ray crystallographic structures of PGIS are available, but those for TXAS are not. The use of homology modeling of the TXAS structure with ns−μs explicit water solvation MD simulations allows much more accurate estimation of the configuration space with loop motion and origin of the protein behaviors in solution. In contrast to the stability of the conserved PGIS structure in solution, the pronounced TXAS flexibility has been revealed to have unstructured loop regions in connection with the characteristic P450 structural elements. The MD-derived and experimental-solution SAXS results are in excellent agreement. The significant protein internal motions, whole-molecule structures, and potential problems with protein folding, crystallization, and functionality are examined

Unraveling the Structure of Magic-Size (CdSe)₁₃ Cluster Pairs

Cadmium selenide is a II–VI semiconductor model system known for its nanoparticle preparation, growth mechanism, luminescence properties, and quantum confinement studies. For the past 2 decades, various thermodynamically stable “magic-size nanoclusters (MSCs)” of CdSe have been observed, isolated, and theoretically calculated. Nevertheless, none of the proposed structures were experimentally confirmed due to the small crystal domains beyond the diffraction limit. With a combination of nondestructive SAXS, WAXS, XRD, XPS, EXAFS, and MAS NMR techniques, we were able to verify the phase transformation, shape, size dimension, local bonding, and chemical environments of (CdSe)₁₃ nanoclusters, which are indicative of a paired cluster model. These experimental results are consistent with the size, shape, bond lengths, dipole moment, and charge densities of the proposed “paired-tubular geometry” predicted by computational approaches. In this article, we revisit the formation pathway of the mysterious (CdSe)₁₃ nanoclusters and propose a paired cluster structure model for better understanding of II–VI semiconductor nanoclusters

Membrane Charging and Swelling upon Calcium Adsorption as Revealed by Phospholipid Nanodiscs

Direct binding of calcium ions (Ca²⁺) to phospholipid membranes is an unclarified yet critical signaling pathway in diverse Ca²⁺-regulated cellular phenomena. Here, high-pressure-liquid-chromatography, small-angle X-ray scattering (SAXS), UV–vis absorption, and differential refractive index detections are integrated to probe Ca²⁺-binding to the zwitterionic lipid membranes in nanodiscs. The responses of the membranes upon Ca²⁺-binding, in composition and conformation, are quantified through integrated data analysis. The results indicate that Ca²⁺ binds specifically into the phospholipid headgroup zone, resulting in membrane charging and membrane swelling, with a saturated Ca²⁺-lipid binding ratio of 1:8. A Ca²⁺-binding isotherm to the nanodisc is further established and yields an unexpectedly high binding constant <i>K</i> = 4260 M^–1 and a leaflet potential of ca. 100 mV based on a modified Gouy–Chapman model. The calcium-lipid binding ratio, however, drops to 40% when the nanodisc undergoes a gel-to-fluid phase transition, leading to an effective charge capacity of a few μF/cm²

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