Role of Macromolecular Crowding on Stability and Iron Release Kinetics of Serum Transferrin

Abstract

The macromolecular crowding influences the structural stability and functional properties of transferrin (Tf). The equilibrium as well as kinetic studies of Tf at different concentrations of crowding agents (dextran 40, dextran 70, and ficoll 70) and at a fixed concentration of dextran 40 under different concentrations of NaCl at pH 7.4 and 5.6 (±1) revealed that (i) the crowder environment increases the diferric-Tf (FeTf) stability against iron loss and overall denaturation of the protein, (ii) both in the absence and presence of crowder, the presence of salt promotes the loss of iron and overall denaturation of FeTf which is due to ionic screening of electrostatic interactions, (iii) the crowder environment retards iron release from monoferric N-lobe of Tf (FeTf) by increasing enthalpic barrier, (iv) the retardation of iron release by crowding is enthalpically dominated than the entropic one, (v) both in the absence and presence of crowder, the presence of salt accelerates the iron release from FeTf due to ionic screening of electrostatic interactions and anion binding to KISAB sites, and (vi) the crowders environment is unable to diminish (a) the salt-induced destabilization of FeTf against the loss of iron and overall denaturation and (b) the anion effect and ionic screening of diffusive counterions responsible to promote iron release from FeTf.

Associated Water Dynamics Might Be a Key Factor Affecting Protein Stability in the Crowded Milieu

“…Presently, the general consensus is that every biological phenomenon (ca., stability, dynamics, activity, folding, aggregation, association, binding, cell culture, tissue engineering, etc.) is modulated in a crowded environment. − Cell extract should be the best candidate to assess the crowding effect. However, doing an experiment and analyzing the result in such a complex entity is challenging .…”

Crowding and Confinement Effects in Different Polymer Concentration Regimes and Their Roles in Regulating the Growth of Nanotubes

“…The cell interior is crowded with a high content (up to 400 g/L) of macromolecules. , The dense medium is able to modulate the structure and reaction rates of biomolecules such as proteins − and nucleic acids. − If the medium is inert to the solute biomolecules, the effects are attributed to macromolecular crowding and macromolecular confinement, both of which are excluded volume effects dominated by entropy. , The former favors products with a more compact conformation, while the latter tends to befit the shape and size of products to that of the confinement volume. − Recent studies show that the medium can also interact with the solute molecules via nonspecific interactions of enthalpic nature. − Such enthalpic contributions can influence the excluded volume effect of macromolecular crowding and confinement depending on the propensity of those nonspecific interactions.…”

Macromolecular crowding: how shape and interaction affect the structure, function, conformational dynamics and relative domain movement of a multi-domain protein

“…4–26 At present, the general consensus is that every biological phenomenon (like stability, dynamics, activity, folding, aggregation, association, binding, cell culture, tissue engineering, and so on) is modulated in a crowded environment. 4–27 Given that performing an experiment and data analysis in a living cell or cell lysate is extremely challenging, most of the studies involve small co-solutes (that probably represent natural osmolytes) and large synthetic macromolecules (like dextran, Ficoll, and polyethylene glycol (PEG) that represents bio-macromolecules like proteins and nucleic acids) as the crowding agents. 4–27 Though natural proteins could be a better candidate and physiologically more relevant as a macromolecular crowder, they cannot be used in a systematic study ( ca.…”

“…4–27 Given that performing an experiment and data analysis in a living cell or cell lysate is extremely challenging, most of the studies involve small co-solutes (that probably represent natural osmolytes) and large synthetic macromolecules (like dextran, Ficoll, and polyethylene glycol (PEG) that represents bio-macromolecules like proteins and nucleic acids) as the crowding agents. 4–27 Though natural proteins could be a better candidate and physiologically more relevant as a macromolecular crowder, they cannot be used in a systematic study ( ca. size/shape effect) to decipher the mechanism.…”