A mechanistic examination of salting out in protein-polymer membrane interactions.

Developing a mechanistic understanding of protein dynamics and conformational changes at polymer interfaces is critical for a range of processes including industrial protein separations. Salting out is one example of a procedure that is ubiquitous in protein separations yet is optimized empirically because there is no mechanistic description of the underlying interactions that would allow predictive modeling. Here, we investigate peak narrowing in a model transferrin-nylon system under salting out conditions using a combination of single-molecule tracking and ensemble separations. Distinct surface transport modes and protein conformational changes at the negatively charged nylon interface are quantified as a function of salt concentration. Single-molecule kinetics relate macroscale improvements in chromatographic peak broadening with microscale distributions of surface interaction mechanisms such as continuous-time random walks and simple adsorption-desorption. Monte Carlo simulations underpinned by the stochastic theory of chromatography are performed using kinetic data extracted from single-molecule observations. Simulations agree with experiment, revealing a decrease in peak broadening as the salt concentration increases. The results suggest that chemical modifications to membranes that decrease the probability of surface random walks could reduce peak broadening in full-scale protein separations. More broadly, this work represents a proof of concept for combining single-molecule experiments and a mechanistic theory to improve costly and time-consuming empirical methods of optimization.

Single-Molecule Dynamics Reflect IgG Conformational Changes Associated with Ion-Exchange Chromatography.

Conformational changes of antibodies and other biologics can decrease the effectiveness of pharmaceutical separations. Hence, a detailed mechanistic picture of antibody-stationary phase interactions that occur during ion-exchange chromatography (IEX) can provide critical insights. This work examines antibody conformational changes and how they perturb antibody motion and affect ensemble elution profiles. We combine IEX, three-dimensional single-protein tracking, and circular dichroism spectroscopy to investigate conformational changes of a model antibody, immunoglobulin G (IgG), as it interacts with the stationary phase as a function of salt conditions. The results indicate that the absence of salt enhances electrostatic attraction between IgG and the stationary phase, promotes surface-induced unfolding, slows IgG motion, and decreases elution from the column. Our results reveal previously unreported details of antibody structural changes and their influence on macroscale elution profiles.

Polymer Free Volume Effects on Protein Dynamics in Polystyrene Revealed by Single-Molecule Spectroscopy.

Protein-polymer interactions are critical to applications ranging from biomedical devices to chromatographic separations. The mechanistic relationship between the microstructure of polymer chains and protein interactions is challenging to quantify and not well studied. Here, single-molecule microscopy is used to compare the dynamics of two model proteins, α-lactalbumin and lysozyme, at the interface of uncharged polystyrene with varied molecular weights. The two proteins exhibit different surface interaction mechanisms despite having a similar size and structure. α-Lactalbumin exhibits interfacial adsorption-desorption with residence times that depend on polymer molecular weight. Lysozyme undergoes a continuous time random walk at the polystyrene surface with residence times that also depend on the molecular weight of polystyrene. Single-molecule observables suggest that the hindered continuous time random walk dynamics displayed by lysozyme are determined by the polystyrene free volume, a finding supported by thermal annealing and solvent quality studies. Hindered dynamics are dominated by short-range hydrophobic interactions where the contributions of electrostatic forces are negligible. This work establishes a relationship between the microscale structure (i.e., free volume) of polystyrene polymer chains to nanoscale interfacial protein dynamics.

Generalized method to design phase masks for 3D super-resolution microscopy.

Point spread function (PSF) engineering by phase modulation is a novel approach to three-dimensional (3D) super-resolution microscopy, with different point spread functions being proposed for specific applications. It is often not easy to achieve the desired shape of engineered point spread functions because it is challenging to determine the correct phase mask. Additionally, a phase mask can either encode 3D space information or additional time information, but not both simultaneously. A robust algorithm for recovering a phase mask to generate arbitrary point spread functions is needed. In this work, a generalized phase mask design method is introduced by performing an optimization. A stochastic gradient descent algorithm and a Gauss-Newton algorithm are developed and compared for their ability to recover the phase masks for previously reported point spread functions. The new Gauss-Newton algorithm converges to a minimum at much higher speeds. This algorithm is used to design a novel stretching-lobe phase mask to encode temporal and 3D spatial information simultaneously. The stretching-lobe phase mask and other masks are fabricated in-house for proof-of-concept using multi-level light lithography and an optimized commercially sourced stretching-lobe phase mask (PM) is validated experimentally to encode 3D spatial and temporal information. The algorithms' generalizability is further demonstrated by generating a phase mask that comprises four different letters at different depths.

Enhancing Analytical Separations Using Super-Resolution Microscopy.

Super-resolution microscopy is becoming an invaluable tool to investigate structure and dynamics driving protein interactions at interfaces. In this review, we highlight the applications of super-resolution microscopy for quantifying the physics and chemistry that occur between target proteins and stationary-phase supports during chromatographic separations. Our discussion concentrates on the newfound ability of super-resolved single-protein spectroscopy to inform theoretical parameters via quantification of adsorption-desorption dynamics, protein unfolding, and nanoconfined transport.

Super-Temporal-Resolved Microscopy Reveals Multistep Desorption Kinetics of α-Lactalbumin from Nylon.

Insight into the mechanisms driving protein-polymer interactions is constantly improving due to advances in experimental and computational methods. In this study, we used super-temporal-resolved microscopy (STReM) to study the interfacial kinetics of a globular protein, α-lactalbumin (α-LA), adsorbing at the water-nylon 6,6 interface. The improved temporal resolution of STReM revealed that residence time distributions involve an additional step in the desorption process. Increasing the ionic strength in the bulk solution accelerated the desorption rate of α-LA, attributed to adsorption-induced conformational changes. Ensemble circular dichroism measurements were used to support a consecutive reaction mechanism. Without the improved temporal resolution of STReM, the desorption intermediate was not resolvable, highlighting both STReM's potential to uncover new kinetic mechanisms and the continuing need to push for better time and space resolution.

Variable Lysozyme Transport Dynamics on Oxidatively Functionalized Polystyrene Films.

Tuning protein adsorption dynamics at polymeric interfaces is of great interest to many biomedical and material applications. Functionalization of polymer surfaces is a common method to introduce application-specific surface chemistries to a polymer interface. In this work, single-molecule fluorescence microscopy is utilized to determine the adsorption dynamics of lysozyme, a well-studied antibacterial protein, at the interface of polystyrene oxidized via UV exposure and oxygen plasma and functionalized by ligand grafting to produce varying degrees of surface hydrophilicity, surface roughness, and induced oxygen content. Single-molecule tracking indicates lysozyme loading capacities, and surface mobility at the polymer interface is hindered as a result of all functionalization techniques. Adsorption dynamics of lysozyme depend on the extent and the specificity of the oxygen functionalities introduced to the polystyrene surface. Hindered adsorption and mobility are dominated by hydrophobic effects attributed to water hydration layer formation at the functionalized polystyrene surfaces.

Unraveling peak asymmetry in chromatography through stochastic theory powered Monte Carlo simulations.

An overarching theory of chromatography capable of modeling all analyte-stationary phase interactions would enable predictive design of pharmaceutically relevant separations. The stochastic theory of chromatography has been postulated as a suitable basis to achieve this goal. Here, we implement Dondi and Cavazzini's Monte Carlo framework that utilizes experimentally accessible single molecule kinetics and use it to correlate heterogenous adsorption statistics at the stationary phase to shifts in asymmetry. The contributions cannot be captured or modeled through ensemble chemometrics. Simulations reveal that peak asymmetry scales non-linearly with longer analyte-stationary phase interactions and migrates towards symmetry across the column length, even without column overloading.

Super Temporal-Resolved Microscopy (STReM).

Super-resolution microscopy typically achieves high spatial resolution, but the temporal resolution remains low. We report super temporal-resolved microscopy (STReM) to improve the temporal resolution of 2D super-resolution microscopy by a factor of 20 compared to that of the traditional camera-limited frame rate. This is achieved by rotating a phase mask in the Fourier plane during data acquisition and then recovering the temporal information by fitting the point spread function (PSF) orientations. The feasibility of this technique is verified with both simulated and experimental 2D adsorption/desorption and 2D emitter transport. When STReM is applied to measure protein adsorption at a glass surface, previously unseen dynamics are revealed.

Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions.

Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.