Size-dependent macromolecular crowding effect on the thermodynamics of protein unfolding revealed at the single molecular level Nilimesh Das, Pratik Sen ⁎ Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208 016, UP, India abstract article info Article history: Received 9 July 2019 Received in revised form 30 August 2019 Accepted 4 September 2019 Available online 06 September 2019 The real biological environment involves a high degree of complexity and the macromolecular crowder is the best candidate to somewhat mimic this. In this contribution, we have used two different sized dextrans as model crowders and human serum albumin (HSA) as a model protein to decipher how the thermal stability of protein is modulated inside the crowded milieu and also to understand the effect of the size of the crowders. In our previous report (Biochemistry 2018, 57, 6078–6089) we have proposed the presence of some interaction between dextran-6 and HSA, which are probably not present between the larger dextrans and HSA. Complete thermodynamic analysis of thermal denaturation profile of HSA suggests that small crowders increase protein stability mainly via the enthalpy of denaturation while larger crowders increase stability primarily through en- tropy. Further, the active site dynamics is altered significantly in the presence of larger dextran-40, but not by smaller dextran-6. Surprisingly, the dynamics of the more compact intermediate state does not get modified by the crowders. Overall, our result indicates that biomacromolecules of similar chemical composition and shape may exert their effect not only by different extent but also by a different mechanism, owing to their differ- ent sizes. © 2019 Elsevier B.V. All rights reserved. Keywords: Macromolecular crowding Size effect Protein unfolding Conformational fluctuation dynamics Thermal stability 1. Introduction Cell environment is exceptionally complex with the presence of pro- teins, sugars, osmolytes, nucleic acids, amino acids, molecular chap- erons, inorganic salts, which are commonly known as crowders [1,2]. Depending on the size of the crowders, they are sub-classified as molec- ular or macromolecular crowders. It has been realized that these crowders could have a profound effect on the intracellular reactions through specific and non-specific interactions [3,4]. For example, the enzymatic activity or the structure of the active form of a protein in the actual cellular environment may be very different in comparison to the bulk buffer, where these crowders are absent [5–7]. These crowder molecules occupy a large cellular volume (almost 30–40% w/ v) and exclude some volume within the cell to be accessed by others [8,9]. Moreover because of the presence of the macromolecules in such high concentration, the solution inside the cell is 3–4 times more viscous than the buffer solution, usually used in in vitro studies [10]. All these will have a profound effect on the structure, dynamics, activity, stability, aggregation behavior and folding-unfolding kinetics of a pro- tein [11–19]. The polymer looping is also found to be controlled by macromolecular crowding [20–22]. However, the in-cell crowding in- vestigation or even the in-vitro cell mimicking using cell extracts are very difficult because of extremely complex and heterogeneous nature of the cellular interior. In such environment, the modulation of the pro- tein signal might originate not only due to the bulk-crowding, but also from micro-environmental difference, confinement, adsorption, phase separation, etc. [23–26]. Instead, a varieties of synthetic substances, ca. polyethylene glycols (PEG), dextrans, ficolls, are usually used as the mo- lecular crowders. In this investigation, we have focused on the stability of a protein in- side the crowded milieu using artificial macromolecules (dextrans). Protein stability is generally characterized as the thermodynamic stabil- ity of the protein. This is to note that proteins are the most fundamental biomolecule inside a living body, and perform the essential molecular reactions in an extremely complicated fashion [27,28]. In such circum- stances, different types of forces on the protein decides in which form it will stay within the cell [29]. Various factors can be responsible for al- tering this particular form of the protein and the ability of the protein to retain its original structural parameters against this disruption is called its stability [30]. The more a protein can maintain its specific structural motif against the disrupting forces, the more is its stability. Protein func- tions depend strongly on its structural parameters and hence on its sta- bility. Therefore, the study of protein stability remains an exciting field International Journal of Biological Macromolecules 141 (2019) 843–854 ⁎ Corresponding author at: Department of Chemistry, IIT Kanpur, India. E-mail address: psen@iitk.ac.in (P. Sen). https://doi.org/10.1016/j.ijbiomac.2019.09.029 0141-8130/© 2019 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac