Challenges for design of aggregation-resistant variants of granulocyte colony-stimulating factor.

Non-native protein aggregation is a long-standing issue in pharmaceutical biotechnology. A rational design approach was used in order to identify variants of recombinant human granulocyte colony-stimulating factor (rhG-CSF) with lower aggregation propensity at solution conditions that are typical of commercial formulation. The approach used aggregation-prone-region (APR) predictors to select single amino acid substitutions that were predicted to decrease intrinsic aggregation propensity (IAP). The results of static light scattering temperature-ramps and chemical unfolding experiments demonstrated that none of the selected variants exhibited improved aggregation resistance, and the apparent conformational stability of each variant was lower than that of WT. Aggregation studies under partly denaturing conditions suggested that the IAP of at least one variant remained unaltered. Overall, this study highlights a general challenge in designing aggregation resistance for proteins, due to the need to accurately predict both APRs and conformational stability.

Driving Forces for Nonnative Protein Aggregation and Approaches to Predict Aggregation-Prone Regions.

Nonnative protein aggregation is the process by which otherwise folded, monomeric proteins are converted to stable aggregates composed of protein chains that have undergone some degree of unfolding. Often, a conformational change is needed to allow certain sequences of amino acids-so-called aggregation-prone regions (APRs)-to form stable interprotein contacts such as ß-sheet structures. In addition to APRs that are needed to stabilize aggregates, other factors or driving forces are also important in inducing aggregation in practice. This review focuses first on the overall process and mechanistic drivers for nonnative aggregation, followed by a more detailed summary of the factors currently thought to be important for determining which amino acid sequences most greatly stabilize nonnative protein aggregates, as well as a survey of many of the existing algorithms that are publicly available to attempt to predict APRs. Challenges with experimental validation of predicted APRs for proteins are briefly discussed.

Diffusing colloidal probes of protein-carbohydrate interactions.

We present diffusing colloidal probe measurements of weak, multivalent, specific protein-polysaccharide interactions mediated by a competing monosaccharide. Specifically, we used integrated evanescent wave and video microscopy methods to monitor the three-dimensional Brownian excursions of conconavilin A (ConA) decorated colloids interacting with dextran-functionalized surfaces in the presence of glucose. Particle trajectories were interpreted as binding lifetime histograms, binding isotherms, and potentials of mean force. Binding lifetimes and isotherms showed clear trends of decreasing ConA-dextran-specific binding with increasing glucose concentration, consistent with expectations. Net potentials were accurately captured by superposition of a short-range, glucose-independent ConA-dextran repulsion and a longer-range, glucose-dependent dextran bridging attraction modeled as a harmonic potential. For glucose concentrations greater than 100 mM, the net ConA-dextran potential was found to have only a nonspecific repulsion, similar to that of bovine serum albumin (BSA) decorated colloids over dextran determined in control experiments. Our results demonstrate the first use of optical microscopy methods to quantify the connections between potentials of mean force and the binding behavior of ConA-decorated colloids on dextran-functionalized surfaces.

Imaging carbon nanotube interactions, diffusion, and stability in nanopores.

We report optical microscopy measurements of three-dimensional trajectories of individual multiwalled carbon nanotubes (MWCNTs) in nanoscale silica slit pores. Trajectories are analyzed to nonintrusively measure MWCNT interactions, diffusion, and stability as a function of pH and ionic strength. Evanescent wave scattering is used to track MWCNT positions normal to pore walls with nanometer-scale resolution, and video microscopy is used to track lateral positions with spatial resolution comparable to the diffraction limit. Analysis of MWCNT excursions normal to pore walls yields particle-wall potentials that agree with theoretical electrostatic and van der Waals potentials assuming a rotationally averaged potential of mean force. MWCNT lateral mean square displacements are used to quantify translational diffusivities, which are comparable to predictions based on the best available theories. Finally, measured MWCNT pH and ionic strength dependent stabilities are in excellent agreement with predictions. Our findings demonstrate novel measurement and modeling tools to understand the behavior of confined MWCNTs relevant to a broad range of applications.