Particle-based MR modeling with diffusion, microstructure, and enzymatic reactions.

PURPOSE: To develop a novel particle-based in silico MR model and demonstrate applications of this model to signal mechanisms which are affected by the spatial organization of particles, including metabolic reaction kinetics, microstructural effects on diffusion, and radiofrequency (RF) refocusing effects in gradient-echo sequences. METHODS: The model was developed by integrating a forward solution of the Bloch equations with a Brownian dynamics simulator. Simulation configurations were then designed to model MR signal dynamics of interest, with a primary focus on hyperpolarized 13C MRI methods. Phantom scans and spectrophotometric assays were conducted to validate model results in vitro. RESULTS: The model accurately reproduced the reaction kinetics of enzyme-mediated conversion of pyruvate to lactate. When varying proportions of restrictive structure were added to the reaction volume, nonlinear changes in the reaction rate measured in vitro were replicated in silico. Modeling of RF refocusing effects characterized the degree of diffusion-weighted contribution from preserved residual magnetization in nonspoiled gradient-echo sequences. CONCLUSIONS: These results show accurate reproduction of a range of MR signal mechanisms, establishing the model's capability to investigate the multifactorial signal dynamics such as those underlying hyperpolarized 13C MRI data.

Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Structure of the Functionally Important Extracellular Loop C of Human Aquaporin 1 Obtained by Solid-State NMR under Nearly Physiological Conditions.

Human aquaporin 1 (hAQP1) is the first discovered selective water channel present in lipid membranes of multiple types of cells. Several structures of hAQP1 and its bovine homolog have been obtained by electron microscopy and X-ray crystallography, giving a consistent picture of the transmembrane domain with the water-conducting pore. The transmembrane domain is formed by six full helices and two half-helices, which form a central constriction with conserved asparagine-proline-alanine motifs. Another constriction, the aromatic/arginine (ar/R) filter, is found close to the extracellular surface, and includes aromatic residues and a conserved arginine (Arg-195). Although the existing crystal structures largely converge on the location of helical segments, they differ in details of conformation of the longest extracellular loop C and its interactions with the ar/R filter (in particular, with Arg-195). Here, we use solid-state nuclear magnetic resonance to determine multiple interatomic distances, and come up with a refined structural model for hAQP1, which represents a physiologically relevant state predominant at noncryogenic temperatures in a lipid environment. The model clearly disambiguates the position of the Arg-195 sidechain disputed previously and shows a number of interactions for loop C, both with the ar/R filter and a number of other residues on the extracellular side of hAQP1.