Introduction

Deep Mutation Scanning (DMS)

The function of a protein is highly influenced by its three-dimensional structure. It is commonly known that proteins can adopt multiple conformations, each corresponding with unique functions and interactions within the cellular environment. However, a protein can also undergo misfolding, where it's alternative strucutres impact cellular homeostasis. These alternative structures have been implicated in diseases such as Alzheimer's and Parkinson's [1]. The factors that cause a protein take up a misfolded structure is almost entirely unknown. Thus, understanding the nature conformational differences within a protein  provide a fundamental understanding of biological function and diseases. 

DMS is a high-throughput assay that allows assessment of the functional characteristics of a large number of variants of a protein simultaneously in living cells [2]. The technique systematically maps the sequence-activity landscape of proteins under controlled experimental conditions [3]. Given a protein of interest, an exhaustive library of missense variants is formed. Every protein has one amino acid substitution at any given point ultimately accounting for all potential variations. The library is screened for an activity of interest, and the activity of each variant is quantified through deep sequencing. The pattern of mutational impact on activity can be leveraged to build structural models of proteins, as well as suggest the conformation(s) responsible for the activity of interest (figure 1). 

α-Synuclein

α-syn is an example of an intrinsically disordered protein that can misfold to take up multiple amyloid structures. Despite the extensive research already performed on α-syn, these conformational states are still relatively undefined in their prevalence and impact in cells. α-syn has been implicated in diseases such as Parkinson's disease, multiple system atrophy (MSA), and dementia with Lewy bodies (DLP) [4]. However, the exact mechanisms remain a mystery. 

It is known that α-syn conformations have unique characteristics, structural behavior, toxicity, aggregation, and propagation properties, all elements that are influenced by the structure of α-syn is decribed by melki et al [5]. Significantly, it has recently emerged that amyloid structures that arise can cause different disease developments. For instance, distinct polymorphs of α-syn isolated from patients with MSA and DLP had a difference of 1000-fold in cellular toxicity, attributed to the structural and functional differences [4]. By exploring the causes and nature of these conformations, the prion-like propagation of misfolds and subsequent disease pathology can be better understood.

While previous experiments have shown that mutations affect function and toxicity, it was not enough to infer a specific structure. Meanwhile, the data provided by DMS can indicate specific structural conformations based on the differential sensitivity of the yeast to the mutations (figure 2). As such, DMS has been applied to α-syn as a solution to this issue. By generating a variant library of α-syn, transforming it into S. cerevisiae, and producing a fitness landscape, the biologically active conformations of α-syn were defined [3]. By applying different conditions to the library, the relevant α-syn structures can be explored in various cellular contexts [6].