Porous media represent a diverse class of materials in which microstructural features govern key physical, chemical, and biological properties. Magnetic resonance relaxation measurements provide a powerful, nondestructive, approach to study of the pore structure and fluid dynamics in porous media. Such measurements yield valuable information on diffusivity, fluid-solid surface interactions, pore size distributions, and surface relaxivity. However, existing theoretical interpretations of relaxation remain incomplete and sometimes contradictory, particularly in complex porous systems. Brownstein-Tarr (BT) theory is a key framework for understanding magnetic resonance relaxation in porous media. Despite its wide use, the theory is not fully appreciated. Often, fast exchange behaviour is assumed, although it is only one regime of behaviour in Brownstein-Tarr theory. This review provides a brief outline of Brownstein-Tarr theory and its applications in porous media. Interpretation of magnetic resonance (MR) measurements with Brownstein-Tarr theory relies on identifying the specific relaxation-diffusion regimes. Diffusivity plays different roles across distinct regimes, particularly in response to temperature change. However, the influence of diffusivity on observed relaxation lifetimes has rarely been considered. More recent studies that emphasize the effect of diffusivity on relaxation have advanced our understanding of Brownstein-Tarr theory and relaxation behaviour in porous media.
Far from being static structures, it is now well accepted that proteins are highly dynamic entities even in the solid state. Here, we review recent progress in application of magic-angle spinning (MAS) NMR for studying the site-specific dynamics of proteins, as assessed using spin-relaxation and measurement of anisotropic spin interactions. We focus on the types of experimental data that are available, and how best to access the information which is provided by these experimental measurements; in particular, we provide a retrospective on the field in light of recent advances in analytical methodologies, which have led to a slow paradigm shift in how we interpret relaxation rate constant measurements made in the solid state.
Biomolecular condensates formed by liquid–liquid phase separation are inherently biphasic systems in which a condensed phase coexists with a surrounding dilute phase that contains vastly different solute concentrations. While NMR spectroscopy offers unique, quantitative access to molecular structure, interactions and dynamics, its application to phase-separated systems has long been challenged by signal overlap, slow diffusion, and the line broadening in the condensed phase. Recent methodological developments have begun to overcome these limitations by explicitly exploiting the physical properties of biomolecular condensates. In this review, we discuss emerging NMR approaches that enable the characterization of condensates in their native biphasic state. These include the diffusion-based strategy LLPS REDIFINE to disentangle dilute and condensed phase populations and enable quantitative extraction of interphase exchange and droplet properties, the magnetization-transfer-based solvent detection approach CONDENSE-MT, that provides access to NMR-invisible condensed phases, and multiplexed filtering schemes that resolve multiple coexisting components within complex condensates. Together, these methods extend the scope of NMR beyond homogeneous solutions, providing complementary and label-free insights into the structure, dynamics, and phase behavior of multicomponent biomolecular condensates.
Protein self-association and aggregation are common phenomena that occur in various environments, including live cells, research samples, and during bioprocessing or storage of biopharmaceuticals. They may be a part of native biological function, or cause diseases, it can be an artefact in protein research, a concerning phenomenon in biopharmaceutical protein formulation, or a favourable opportunity to spontaneously concentrate proteins. The consequences of protein self-association and aggregation vary across different fields, and therefore may require somewhat different analytical approaches for their assessment and characterization. In this review we focus on types of aggregation and self-association which occur in protein formulations prepared for diverse purposes, where the aggregation itself is not the main functional feature of a protein. We aim to first highlight some major pathways of protein self-interaction and outline the terminology around the process of protein molecules clumping together, and the level of structural changes involved for each major pathway. We will briefly overview various analytical methods for characterising protein aggregation and self-association, and then consider the role of NMR, highlighting NMR parameters that are frequently used to gain insight into these processes. We focus on signal line broadening, chemical shift perturbation, diffusion coefficients, relaxation parameters and spatially selective NMR as the main approaches used to characterise various protein particles and the kinetics of their formation. Lastly, we discuss in more detail a few recent examples of NMR applications to study protein self-association and aggregation mainly in biopharmaceutical context, of how NMR measurables can assist in profiling of higher-order-structure, studies of protein-excipient interactions in formulation development, study of liquid-liquid phase separation and assessment of protein behaviour in complex mixtures. We also discuss low-field NMR methods that are being developed for in-line process monitoring as well as quality control.
