Analysis of Ordered and Disordered Protein Complexes Reveals Structural Features Discriminating Between Stable and Unstable Monomers Kannan Gunasekaran 1 *, Chung-Jung Tsai 1 and Ruth Nussinov 1,2 * 1 Laboratory of Experimental and Computational Biology Basic Research Program SAIC-Frederick, Inc. NCI-Frederick, Frederick MD 21702, USA 2 Department of Human Genetics and Molecular Medicine, Sackler Institute of Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 Israel Most proteins exist in the cell as multi-component assemblies. However, which proteins need to be present simultaneously in order to perform a given function is frequently unknown. The first step toward this goal would be to predict proteins that can function only when in a complexed form. Here, we propose a scheme to distinguish whether the protein components are ordered (stable) or disordered when separated from their complexed partners. We analyze structural characteristics of several types of complexes, such as natively unstructured proteins, ribosomal proteins, two-state and three-state complexes, and crystal-packing dimers. Our analysis makes use of the fact that natively unstructured proteins, which undergo a disorder-to-order transition upon binding their partner, and stable monomeric proteins, which exist as dimers only in their crystal form, provide examples of two vastly different scenarios. We find that ordered monomers can be distinguished from disordered monomers on the basis of the per-residue surface and interface areas, which are significantly smaller for ordered proteins. With this scale, two-state dimers (where the monomers unfold upon dimer separation) and ribosomal proteins are shown to resemble disordered proteins. On the other hand, crystal-packing dimers, whose monomers are stable in solution, fall into the ordered protein category. While there should be a continuum in the distributions, nevertheless, the per-residue scale measures the confidence in the determination of whether a protein can exist as a stable monomer. Further analysis, focusing on the chemical and contact preferences at the interface, interior and exposed surface areas, reveals that disordered proteins lack a strong hydrophobic core and are composed of highly polar surface area. We discuss the implication of our results for de novo design of stable monomeric proteins and peptides. q 2004 Elsevier Ltd. All rights reserved. Keywords: disordered proteins; natively unstructured proteins; protein– protein interactions; folding mechanisms; two-state and three-state complexes *Corresponding authors Introduction A large number of proteins perform their biological functions as oligomers, consisting of two or more polypeptide chains. The underlying principle of protein–protein association has been the subject of many investigations. 1,2 Recognizing proteins that function only as oligomers is crucial to the understanding of protein networking, function and malfunction. 3–5 Many attempts have been made to understand the folding mechanism of oligomers, specifically dimers, based on energetic arguments, and surface and interface character- istics. 5–9 Efforts have focused on distinguishing between specific and non-specific binding charac- teristics of multimers. 10–14 Non-specific contacts between neighboring molecules in crystals are meaningless from the functional standpoint, and are considered “artifacts” of crystallization. Never- theless, the differentiation of functional protein– protein interfaces from those of crystal-packing 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. Abbreviations used: ASA, solvent-accessible surface area; SAPs, structurally ambivalent peptides; PDB, Protein Data Bank. E-mail addresses of the corresponding authors: guna@ncifcrf.gov; ruthn@ncifcrf.gov doi:10.1016/j.jmb.2004.07.002 J. Mol. Biol. (2004) 341, 1327–1341