Unusual domain movement in a multidomain protein in the presence of macromolecular crowders

Abstract

Domain movements play a fundamental and critical role in the specific biological function that multidomain proteins have evolved to perform. A significant amount of research has been carried out to investigate the effects of macromolecular crowding agents, mostly on single domain proteins, thereby furthering our appreciation for the crowding phenomenon. However similar studies on proteins having multiple domains are relatively scarce. Using the plasma protein human serum albumin (HSA) as the protein of interest, we have probed the influence of dextran based crowding agents (Dextran 6, Dextran 40, and Dextran 70) on the relative movements of domains I and II using FRET, with Trp-214 in domain II acting as the donor and acrylodan (Ac) covalently attached to Cys-34 of domain I as the acceptor. Amongst the higher molecular weight crowders, while both Dextran 70 and Dextran 40 induced a significant decrease in the distance between the aforesaid domains, however for the latter macromolecular crowder (Dextran 40), beyond 50 g L(-1), no change in domain separation was observed even up to concentrations of 175 g L(-1). On the other hand, contrary to our expectations, Dextran 6, having the highest packing density by virtue of it being the smallest crowding agent used, provided an asymmetric excluded volume which resulted in forced elongation of HSA along the Trp-Ac FRET axis. Additionally both chemical and thermal studies performed at varying concentrations of the chemical denaturant, urea, reveal unusual movements of the two domains, an aspect that can have important implications with regard to HSA being an avid transporter of fatty acids, with the binding of latter being known to invoke appreciable domain displacements. We hypothesise that we see a distinct crossover from entropy dominated depletion effects in the case of Dextran 6 to significant enthalpic contribution for Dextran 70 with Dextran 40 lying midway between these two crowders, having characteristics of both.

Structural, Functional, and Dynamical Responses of a Protein in a Restricted Environment Imposed by Macromolecular Crowding

“…39,40 On the other hand, for dextran-6, there was a surprising increase in the distance between domain-I and domain-II. 39 In another contribution, they reported that the solvation dynamics of domain-I of HSA depends highly on the concentration of the crowder, owing mainly to the stiffness of the protein matrix. 41 In a recent article, Samanta et al concluded that in the presence of polyethylene glycol (PEG), another type of popular crowder, the α-helicity of HSA increases to a considerable extent.…”

“…HSA is the most abundant transport protein in the human body. , It is a large multidomain protein with three distinct domains, namely, domains I, II, and III . All of these domains are responsible for binding and transporting different classes of molecules like drugs, enzymes, hormones, carbohydrates, etc. − There are several reports on the behavior of HSA in the crowded environment in recent years. − Singh et al monitored the tryptophan fluorescence intensity of HSA as a function of the extent of crowding . They have concluded that the quenching of tryptophan fluorescence by a crowder is mainly static in nature and that dextran-6 exhibits maximum quenching owing to its highest excluded volume by dextran-6.…”

“…They have concluded that the quenching of tryptophan fluorescence by a crowder is mainly static in nature and that dextran-6 exhibits maximum quenching owing to its highest excluded volume by dextran-6. Biswas et al have studied the domain movement of HSA through FRET analysis as a function of crowder concentration and observed a gradual decrease of the interdomain distance between domain-I and domain-II and between domain-II and domain-III for dextran-40 and dextran-70. , On the other hand, for dextran-6, there was a surprising increase in the distance between domain-I and domain-II . In another contribution, they reported that the solvation dynamics of domain-I of HSA depends highly on the concentration of the crowder, owing mainly to the stiffness of the protein matrix .…”

Protein Composition Determines the Effect of Crowding on the Properties of Disordered Proteins

“…The presence of crowding macromolecules has been shown to affect various protein processes, including protein-protein interactions, protein aggregation, and protein folding (3)(4)(5)(6). In protein folding, although crowdinginduced changes in the folded state have been reported (7), the observed stabilization of the folded state under crowded conditions is commonly attributed to destabilization of the unfolded state of the protein (8)(9)(10). The primary effect of crowding is the reduction of the volume available to proteins, which results in excluded volume effects (11).…”

Synthesis, Crystal Structures and Binding Studies of Flufenamic‐ Acid‐Based Metal Complexes

“…Serum proteins are the most abundant proteins present in the blood plasma accounting for over 50% of total protein content . Human serum albumin (HSA) being the avid transporter of fatty acids, different exogenous and endogenous drug molecules, it is important to study the interaction of the compounds with the serum proteins. In this work we examined the interactions of the solids 1–4 with bovine serum albumin (BSA) which has 67% of sequence homology to HSA .…”