The effect of charged residue substitutions on the thermodynamics of protein-surface interactions.

The interactions of proteins with surfaces are important in both biological processes and biotechnologies. In contrast to decades of study regarding the biophysics of proteins in bulk solution, however, our mechanistic understanding of the biophysics of proteins interacting with surfaces remains largely qualitative. In response, we have set to explore quantitatively the thermodynamics of protein-surface interactions. In this work, we explore systematically the role of electrostatics in modulating the interaction between proteins and charged surfaces. In particular, we use electrochemistry to explore the extent to which a macroscopic, hydroxyl-coated surface held at a slightly negative potential affects the folding thermodynamics of surface-attached protein variants with different composition of charged amino acids. Doing so, we find that attachment to the surface generally leads to a net stabilization, presumably due to excluded volume effects that reduce the entropy of the unfolded state. The magnitude of this stabilization, however, is strongly correlated with the charged-residue content of the protein. In particular, we find statistically significant correlations with both the net charge of the protein, with greater negative charge leading to less stabilization by the surface, and with the number of arginines, with more arginines leading to greater stabilization. Such findings refine our understanding of protein-surface interactions, providing in turn a guiding rationale to achieve the functional deposition of proteins on artificial surfaces for implementation in, for example, protein-based biotechnologies.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

Attachment of Proteins to a Hydroxyl-Terminated Surface Eliminates the Stabilizing Effects of Polyols.

The physics of proteins interacting with surfaces can differ significantly from those seen when the same proteins are free in bulk solution. As an example, we describe here the extent to which site-specific attachment to a chemically well-defined macroscopic surface alters the ability of several stabilizing and destabilizing cosolutes to modulate protein folding thermodynamics. We determined this via guanidinium denaturations performed in the presence of varying concentrations of cosolutes when proteins were either site-specifically attached to self-assembled monolayers on gold or free in bulk solution. Doing this we found that the extent to which guanidinium (a destabilizing Hofmeister cation), sulfate (a stabilizing Hofmeister anion), and urea (a neutral denaturant) alter the folding free energy remains indistinguishable whether proteins are surface-attached or free in bulk solution. In sharp contrast, however, neutral osmolytes sucrose and glycerol, which significantly stabilize proteins in bulk solution, do not measurably affect their stability when they are attached to a hydroxyl-terminated surface. In contrast, we recovered bulk solution-like stabilization when the attachment surface was instead carboxyl-terminated. It thus appears that chemistry-specific surface interactions can dramatically alter the way in which biomolecules interact with other components of the system.