Protein−Protein Interactions Affect Alpha Helix Stability in Crowded Environments Bryanne Macdonald, † Shannon McCarley, † Sundus Noeen, † and Alan E. van Giessen* Department of Chemistry, Mount Holyoke College, 50 College Street, South Hadley, Massachusetts 01075, United States *S Supporting Information ABSTRACT: The dense, heterogeneous cellular environment is known to affect protein stability through interactions with other biomacromo- lecules. The effect of excluded volume due to these biomolecules, also known as crowding agents, on a protein of interest, or test protein, has long been known to increase the stability of a test protein. Recently, it has been recognized that attractive protein−crowder interactions play an important role. These interactions affect protein stability and can destabilize the test protein. However, most computational work investigating the role of attractive interactions has used spherical crowding agents and has neglected the specific roles of crowding agent hydrophobicity and hydrogen bonding. Here we use multicanonical molecular dynamics and a coarse-grained protein model to study the folding thermodynamics of a small helical test protein in the presence of crowding agents that are themselves proteins. Our results show that the stability of the test protein depends on the hydrophobicity of the crowding agents. For low values of crowding agent hydrophobicity, the excluded volume effect is dominant, and the test protein is stabilized relative to the dilute solution. For intermediate values of the crowding agent hydrophobicity, the test protein is destabilized by favorable side chain−side chain interactions stabilizing the unfolded states. For high values of the crowding agent hydrophobicity, the native state is stabilized by the strong intermolecular attractions, causing the formation of a packed structure that increases the stability of the test protein through favorable side chain−side chain interactions. In addition, increasing crowding agent hydrophobicity increases the “foldability” of the test protein and alters the potential energy landscape by simultaneously deepening the basins corresponding to the folded and unfolded states and increasing the energy barrier between them. ■ INTRODUCTION The cellular environment in which proteins fold and function is crowded with biomacromolecules and is known to affect protein stability relative to dilute solution. 1 Much of the work investigating the effect of a crowded environment on the stability of proteins has focused on macromolecular crowding by using large, inert macromolecules to reproduce the crowded cellular milieu. 2 Experimental investigations of macromolecular crowding frequently use large, neutral, and inert molecules such as Ficoll or dextran as crowding agents. 3 In theoretical and computational studies, these are usually represented as inert spheres that have no interactions with the protein other than excluded volume interactions. 4 It is now widely accepted that it is essential to include the attractive interactions between the protein of interest, the “test protein,” and other cellular macromolecules, referred to as “crowding agents”. 5−11 Of the computational work that includes attractive interactions between the test protein and crowding agents, most continue to use large unphysical spheres as crowding agents, with either single 7,12 or multiple 13 interaction sites per crowder with a fixed attractive interaction. The only work to vary the strength of the attraction between the test protein and the (spherical) crowders 7,12 focuses on protein association and not stability. Recently, efforts have been made to include both attractive interactions and excluded volume effects by studying protein stability in a more realistic cellular-like environment in silico, 9,11,14−19 in vitro, 6,10 and in vivo. 5,20−25 In addition, it has been shown that the hydrophobic nature of a chaperonin cavity can affect the stability of the enclosed protein. 26−28 These studies have shown that the stability of a protein is affected by the chemical nature of its environment, not just by the presence of crowding agents. This fact gains importance when one takes into account the heterogeneity of the cellular environment, in terms of both the molecular composition of the cell and its spatial heterogeneity. The molecular composition of the cytoplasm is not uniform but instead varies at different areas in the cell. 29 A protein that is stable in one region of the cell may be unstable in another. Introducing chemical specificity by using proteins as crowding agents is a necessary step toward a more accurate representation of the cellular environment. However, there are as yet no systematic Received: December 18, 2014 Revised: January 13, 2015 Article pubs.acs.org/JPCB © XXXX American Chemical Society A DOI: 10.1021/jp512630s J. Phys. Chem. B XXXX, XXX, XXX−XXX