Traditionally, biochemical studies are performed in dilute homogenous solutions, which are very different from the dense mixture of molecules found in cells. Thus, the physiological relevance of these studies is in question. This recognition motivated scientists to formulate the effect of crowded solutions in general, and excluded volume in particular, on biochemical processes. Using polymers or proteins as crowders, it was shown that while crowding tends to significantly enhance the formation of complexes containing many subunits, dimerizations are only mildly affected. Computer simulations, together with experimental evidence, indicate soft interactions and diffusion as critical factors that operate in a concerted manner with excluded volume to modulate protein binding. Yet, these approaches do not truly mimic the cellular environment. In vivo studies may overcome this shortfall. The few studies conducted thus far suggest that in cells, binding and folding occur at rates close to those determined in dilute solutions. Obtaining quantitative biochemical information on reactions inside living cells is currently a main challenge of the field, as the complexity of the intracellular milieu was what motivated crowding research to begin with.

http://absimage.aps.org/image/MAR11/MWS_MAR11-2010-020038.pdf

Formation of protein-complexes in crowded environments: from in vitro to in vivo

Using the transient hot wire and pulsed field gradient nuclear magnetic resonance methods we determined the thermal conductivity and the solvent self-diffusion coefficient (SDC) in aqueous suspensions of quasi-monodisperse spherical silica nanoparticles. The thermal conductivity was found to increase at higher volume fraction of nanoparticles in accordance with the effective medium theory albeit with a smaller slope. On the other hand, the SDC was found to decrease with nanoparticle volume fraction faster than predicted by the effective medium theory. These deviations can be explained by the presence of an interfacial heat-transfer resistance and water retention by the nanoparticles, respectively. We found no evidence for anomalous enhancement in the transport properties of nanofluids reported earlier by other groups.

Heat- and mass-transport in aqueous silica nanofluids

While NMR is the most used analytical method for determining the molecular structure of isolated chemical entities, small compounds as well as macromolecules, its capability of analysing complex mixtures is less known. The advent of Diffusion Ordered SpectroscopY (DOSY) NMR has made diffusion experiments popular, enabling diffusion coefficients to be routinely measured and used to characterize chemical systems in solution. Indeed, since the translational diffusion coefficients of molecular species reflect their effective sizes and shapes, DOSY NMR allows the separation of the chemical entities present in multicomponent systems and, as in all diffusion NMR experiments, provides information on their intermolecular interactions as well as on their size and shape. The main aim of this review is to present an overview of the DOSY NMR mapping and its applications. The paper starts with a brief introduction to pulsed-field gradient (PFG) NMR and then focuses on the methodological procedures that can be used to perform good diffusion data acquisition and to obtain good-quality DOSY maps. The second part describes, through selected literature examples, different applications of DOSY NMR to demonstrate the potential of the method for (i) unravelling the components of complex matrices comprising pharmaceuticals, dietary supplements, foods and beverages, and biological extracts, and (ii) probing intermolecular interactions and evaluating association constants between different hosts and guests, as well as estimating the sizes and molecular weights of molecular species.

Pulsed-field gradient nuclear magnetic resonance measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping

Inorganic/organic hybrid nanosystems have been increasingly developed for their versatility and efficacy at overcoming obstacles not readily surmounted by nonhybridized counterparts. Currently, hybrid nanosystems are implemented for gene therapy, drug delivery, and phototherapy in addition to tissue regeneration, vaccines, antibacterials, biomolecule detection, imaging probes, and theranostics. Though diverse, these nanosystems can be classified according to foundational inorganic/organic components, accessory moieties, and architecture of hybridization. Within this Review, we begin by providing a historical context for the development of biomedical hybrid nanosystems before describing the properties, synthesis, and characterization of their component building blocks. Afterward, we introduce the architectures of hybridization and highlight recent biomedical nanosystem developments by area of application, emphasizing hybrids of distinctive utility and innovation. Finally, we draw attention to ongoing clinical trials before recapping our discussion of hybrid nanosystems and providing a perspective on the future of the field.

Hybrid Nanosystems for Biomedical Applications.

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size.

/pdf/depletion-interactions-modulate-the-binding-between-2qfs1enxgb.pdf

Depletion interactions modulate the binding between disordered proteins in crowded environments

Diffusion NMR studies of macromolecular complex formation, crowding and confinement in soft materials.

The effect of particles on the behavior of polymers in solution is important in a number of important phenomena such as the effect of ``crowding'' proteins in cells, colloid-polymer mixtures, and nanoparticle ``fillers'' in polymer solutions and melts. In this Letter, we study the effect of spherical inert nanoparticles (which we refer to as ``crowders'') on the diffusion coefficient and radius of gyration of polymers in solution using pulsed-field-gradient NMR and small-angle neutron scattering (SANS), respectively. The diffusion coefficients exhibit a plateau below a characteristic polymer concentration, which we identify as the overlap threshold concentration ${c}^{\ensuremath{\star}}$. Above ${c}^{\ensuremath{\star}}$, in a crossover region between the dilute and semidilute regimes, the (long-time) self-diffusion coefficients are found, universally, to decrease exponentially with polymer concentration at all crowder packing fractions, consistent with a structural basis for the long-time dynamics. The radius of gyration obtained from SANS in the crossover regime changes linearly with an increase in polymer concentration, and must be extrapolated to ${c}^{\ensuremath{\star}}$ in order to obtain the radius of gyration of an individual polymer chain. When the polymer radius of gyration and crowder size are comparable, the polymer size is very weakly affected by the presence of crowders, consistent with recent computer simulations. There is significant chain compression, however, when the crowder size is much smaller than the polymer radius gyration.

/pdf/combining-diffusion-nmr-and-small-angle-neutron-scattering-5by6okca52.pdf

Combining Diffusion NMR and Small-Angle Neutron Scattering Enables Precise Measurements of Polymer Chain Compression in a Crowded Environment

We apply pulsed-field-gradient NMR (PFG NMR) technique to measure the translational diffusion for both uncharged and charged polysaccharide (Ficoll70) in water. Analysis of the data indicate that NMR signal attenuation above a certain packing fraction can be adequately fitted with a bi-exponential function. The self-diffusion measurements show also that the Ficoll70, an often-used compact, spherical polysucrose molecule, is itself non-ideal, exhibiting signs of both softness and attractive interactions in the form of a stable suspension consisting of monomers and clusters. Further, we can quantify the fraction of monomer and cluster. This work strengthens the picture of the existence of a bound water layer within and around a porous Ficoll70 particle.

Dynamics and complex formation in charged and uncharged Ficoll70 solutions

We apply pulsed-field-gradient NMR (PFG NMR) technique to measure the translational diffusion for both uncharged and charged polysaccharide (Ficoll70) in water. Analysis of the data indicates that the NMR signal attenuation above a certain packing fraction can be adequately fitted with a bi-exponential function. The self-diffusion measurements also show that the Ficoll70, an often-used compact, spherical polysucrose molecule, is itself nonideal, exhibiting signs of both softness and attractive interactions in the form of a stable suspension consisting of monomers and clusters. Further, we can quantify the fraction of monomers and clusters. This work strengthens the picture of the existence of a bound water layer within and around a porous Ficoll70 particle.

Dynamics and cluster formation in charged and uncharged Ficoll70 solutions

We have examined the effect of crowder particle charge on macromolecular structure, studied via small-angle neutron scattering, and translational dynamics, studied via pulsed-field gradient NMR, in...

Swomitra Palit

Papers

Diffusion NMR studies of macromolecular complex formation, crowding and confinement in soft materials.

Combining Diffusion NMR and Small-Angle Neutron Scattering Enables Precise Measurements of Polymer Chain Compression in a Crowded Environment

Dynamics and complex formation in charged and uncharged Ficoll70 solutions

Dynamics and cluster formation in charged and uncharged Ficoll70 solutions

The effect of crowder charge in a model polymer-colloid system for macromolecular crowding: Polymer structure and dynamics.