Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle

Emily C Hartman,Marco J Lobba,Ester Álvarez-Benedicto,Andrew H Favor,Matthew B Francis,Danielle Tullman-Ercek,Christopher M Jakobson

doi:10.1038/s41467-018-03783-y

Abstract

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes—a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

Highlights

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge
Most quantified fitness landscapes have been determined for enzymes or proteins with a straightforward selection or screen, where fitness is defined as catalytic activity[14,15], binding[16,17], growth[18], or fluorescence[19]
We describe a library generation and single-step selection strategy—termed SyMAPS (Systematic Mutation and Assembled Particle Selection)—to study the structure a selfassembling protein capsid composed of a noninfectious viral structural protein, or virus-like particle (VLP)

Summary

Introduction

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. We report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. We describe a library generation and single-step selection strategy—termed SyMAPS (Systematic Mutation and Assembled Particle Selection)—to study the structure a selfassembling protein capsid composed of a noninfectious viral structural protein, or virus-like particle (VLP). This selection does not rely on infectivity, clinical abundance, or serum stability, and enables experimental characterization of all single amino acid variants of MS2 bacteriophage coat protein (MS2 CP). SyMAPS is a straightforward approach that can be applied more broadly to assess the fitness landscapes of the coat proteins of clinically relevant pathogens, including hepatitis B and human papillomavirus virions[31]

Methods

Results

Conclusion