Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein–substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

Cysteine is commonly used to attach peptides onto gold surfaces. In this work, we show that the inclusion of an additional linker of four residues in length (-PPPPC) of a rigid, hydrophobic nature, is a better choice for forming peptide self-assembled monolayers (SAMs) with well-ordered structure and high surface density. We compare the structure and function of the nonfouling peptide EKEKEKE-PPPPC-Am with EKEKEKE-C-Am. Circular dichroism (CD), attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR), and molecular dynamics (MD) results show that EKEKEKE-PPPPC-Am forms a secondary structure while EKEKEKE-C-Am has a random structure. Surface plasmon resonance (SPR) sensor results show that protein adsorption on EKEKEKE-PPPPC-Am/gold is very low with small variation while protein adsorption on EKEKEKE-C-Am/gold is high with large variation. X-ray photoelectron spectroscopy (XPS) results show that both peptides have strong gold-thiol binding onto a gold surface, indicating that their difference in protein adsorption is due to their assembled structures. Further experimental and simulation studies were performed to show that -PPPPC is a better linker than -PC, -PPC, and -PPPC. Finally, we extend EKEKEKE-PPPPC-Am with the cell-binding sequence RGD and demonstrate control over specific vs. non-specific cell adhesion without using poly (ethylene) glycol (PEG). Adding a functional peptide to the nonfouling EK sequence avoids complex chemistries that are used for its connection to synthetic materials.

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The interactions which govern chemical processes may be broadly categorized into specific interactions, high activity for a certain target molecule, and nonspecific interactions, low activity for all targets. Despite their ubiquity in biology and chemistry, nonspecific interactions are generally overlooked and a fundamental understanding of nonspecific interactions is lacking. Molecular chaperones are large protein complexes which have evolved to resist nonspecific interactions. Their interior surface resists binding to thousands of types of misfolded proteins. Proteins found in the cytoplasm, a crowded environment with many spurious binding targets, are another example. These proteins have evolved high selectivity and stability despite nonspecific interactions. Using structural bioinformatics, we have studied the interiors of molecular chaperones from five species and examined the surface chemistry of 1162 proteins, categorized by if they are present in the cytoplasm or extracellular space. A better understanding of how nature resists nonspecific interactions is key for the chemistry of materials, surfaces, and particles which must remain stable in complex environments. The abundance of amino acids, their interactions, their hydration, and sequence patterns were compared in these two systems, molecular chaperones and proteins surfaces. Striking similarities were found and trends were identified as the system environments became harsher. Peptide based mimics were synthesized to test the conclusions. This, in turn, has led to the design of new stealth compounds and a deeper understanding of nonspecific interactions.