A thermodynamic definition of protein domains

Abstract

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structurebased decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose mvalue is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.protein folding | protein structure | protein architecture | protein parsing D omains are visually arresting protein substructures with an influential history in protein biochemistry (1). These familiar, self-contained structural units were first noticed in some of the earliest solved protein structures (2, 3) and soon came to be recognized as common features of protein architecture (4).Dissecting proteins into their constituent domains provides a simple, intuitive approach to classifying protein structure, a molecular application of the time-honored principle of "carving nature at its joints" (5). Many structure-based computer algorithms have been devised to parse the ever-increasing number of solved proteins into discrete units; a highly abbreviated sample includes (6-13). Today, CATH (14) and SCOP (15) are the two most widely used domain classifications. Both are based on computational algorithms but rely ultimately on the human eye as the final arbiter of domain boundaries.However, seeing can be deceiving. The dependence on visual intuition introduces an unavoidable element of ambiguity into procedures for domain recognition. The most enduring domain definition, "potentially independent, stable folding uni...

Chaperones Rescue Luciferase Folding by Separating Its Domains

“…9, left panel). These results are similar to algorithm predictions (N-terminal domain, start-266; C-terminal domain, 434-end) that consider multidomain proteins as collections of self-contained cooperative units (48). These divisions are also very similar to the divisions determined during the simulated unfolding (Fig.…”

Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping

“…Analysis of crystal structures of GPIbα with platelet-type VWD mutations that increase affinity reveal a potential disruption of conformational preferences for the opposable thumb to favor a 3 10 helix structure or a conserved D 235 -K 237 salt bridge thereby enabling stretching to the extended conformation found in cocrystal complexes with A1 [35], but crystal structures can often be deceiving [36]. GPIbα preferentially interacts with the disordered RCAM A1 conformational state with high-affinity.…”

Structural Origins of Misfolding Propensity in the Platelet Adhesive Von Willebrand Factor A1 Domain

“…The cooperativity ratio (CR ¼ m excised /m in situ ) evaluates the extent to which a fragment acts as an independent folding unit ( Fig. 2 B) (12). In essence, when native tertiary structure contacts are disrupted, a low CR corresponds to a predisposition to unfold, whereas a high CR indicates a propensity to remain folded.…”

“…3). The median is appropriate considering that a minimum of 30 residues was needed to identify a domain in the original SEED algorithm as implemented by Porter and Rose (12). The per-residue median values provide an empirical thermodynamic profile for comparing protein structures.…”

“…To investigate the origins of the observed differences in the unfolding thermodynamics of the A1 and A3 domains, we have employed the structure-energy-equivalenceof-domains (SEED) algorithm (12), which uses residuespecific water / 1M urea-group transfer free energies (13) to define thermodynamic subdomains of protein structures consistent with experimentally determined equilibrium folding intermediates. In initiating the studies to be described, our goal was to resolve regions of the A1 domain structure that contribute to its propensity to unfold to intermediate conformations by using the A3 domain structure as an experimentally verified two-state reference.…”