Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder

Shahid, Sumra; Hasan, Syed Ikramul; Ahmad, Faizan; Hassan, Md. Imtaiyaz; Islam, Asimul

doi:10.3390/biom9090477

Cited by 33 publications

(23 citation statements)

References 125 publications

(215 reference statements)

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“…The extent of stabilization of α-LA by the crowders (ficoll 70, dextran 70, and dextran 40) increases with the increasing concentration of the crowding agents due to the excluded volume effect and the small-sized and rod-shaped crowder, i.e., dextran 40, causing more pronounced stabilization of the protein in comparison with dextran 70 and ficoll 70 [ 118 ]. The structure of the protein in these experiments remains unperturbed.…”

Section: Unfolding Of α-Lactalbumin Caused By Heat and Various Denmentioning

confidence: 99%

α-Lactalbumin, Amazing Calcium-Binding Protein

Permyakov

2020

Biomolecules

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α-Lactalbumin (α-LA) is a small (Mr 14,200), acidic (pI 4–5), Ca2+-binding protein. α-LA is a regulatory component of lactose synthase enzyme system functioning in the lactating mammary gland. The protein possesses a single strong Ca2+-binding site, which can also bind Mg2+, Mn2+, Na+, K+, and some other metal cations. It contains several distinct Zn2+-binding sites. Physical properties of α-LA strongly depend on the occupation of its metal binding sites by metal ions. In the absence of bound metal ions, α-LA is in the molten globule-like state. The binding of metal ions, and especially of Ca2+, increases stability of α-LA against the action of heat, various denaturing agents and proteases, while the binding of Zn2+ to the Ca2+-loaded protein decreases its stability and causes its aggregation. At pH 2, the protein is in the classical molten globule state. α-LA can associate with membranes at neutral or slightly acidic pH at physiological temperatures. Depending on external conditions, α-LA can form amyloid fibrils, amorphous aggregates, nanoparticles, and nanotubes. Some of these aggregated states of α-LA can be used in practical applications such as drug delivery to tissues and organs. α-LA and some of its fragments possess bactericidal and antiviral activities. Complexes of partially unfolded α-LA with oleic acid are cytotoxic to various tumor and bacterial cells. α-LA in the cytotoxic complexes plays a role of a delivery carrier of cytotoxic fatty acid molecules into tumor and bacterial cells across the cell membrane. Perhaps in the future the complexes of α-LA with oleic acid will be used for development of new anti-cancer drugs.

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Section: Unfolding Of α-Lactalbumin Caused By Heat and Various Denmentioning

confidence: 99%

α-Lactalbumin, Amazing Calcium-Binding Protein

Permyakov

2020

Biomolecules

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“…While both computational [ 3 , 4 ] and experimental [ 1 ] approaches agree that crowding shifts thermodynamic equilibria of reactions towards products, the influence of crowding on reaction kinetics, especially those involving enzymes, is not as well understood. Crowding usually decreases the catalytic activity for diffusion-limited reactions due to impeded enzyme-substrate encounters [ [5] , [6] , [7] , [8] ]. For transition-state-limited reactions, though, crowding effects are less predictable due to the complex, often opposing, interplay of excluded volume, viscosity, soft interactions, and hydration effects [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

“…A depletion layer is only observed with crowders larger than the protein of interest and the thickness of this layer is directly correlated with the size of the crowder [ 47 ]. At the same time, the high concentration of crowder beyond the depletion layer lowers the activity of water [ 8 ], creating an osmotic force that dehydrates the protein surface [ 48 ]. Since unfolded proteins have a higher solvent accessible surface areas than the native state, the unfolded form experiences more dehydration, thereby stabilizing the folded protein [ 39 ].…”

Section: Introductionmentioning

confidence: 99%

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

Wilcox

Chung

Slade

2021

Biochemistry and Biophysics Reports

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In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease V max and K m for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in V max is likely due to excluded volume effects, which are partially counteracted by viscosity hindering release of the NAD + product. Macromolecular crowding is further complicated by the presence of a depletion layer in solutions of dextran larger than YADH, which diminishes the hindrance from viscosity. The disparate effects from 25 g/L dextran or glucose compared to 25 g/L Ficoll or sucrose reveals that soft interactions must also be considered. Data from binary mixtures of glucose, dextran, and Ficoll support this “tuning” of opposing factors. While macromolecular crowding was originally proposed to influence proteins mainly through excluded volume effects, this work compliments the growing body of evidence revealing that other factors, such as preferential hydration, chemical interactions, and the presence of a depletion layer also contribute to the overall effect of crowding.

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“…Native cellular environments, such as the cytoplasm, are packed with biomacromolecules that subject proteins to highly confined and crowded environments, and these conditions have been shown to impact the equilibria of biochemical processes such as protein folding, as well as protein function and stability [ 2 , 3 ]. Given that traditional experimental protocols for the study of protein fates involved dilute buffer systems and did not replicate the in vivo conditions, new models have been developed to mimic natural confinement and crowding by using macromolecules, mesoporous silica matrices, reverse micelles, or hydrogels [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 25 ]. These studies demonstrated significant effects on several protein fates, including activity and functional stability, their interactions with self and other proteins, and unfolding and aggregation.…”

Section: Discussionmentioning

confidence: 99%

“…Inside cells, proteins exist and function in highly crowded and compartmentalized environments that have a significant impact on several of their functions, including diffusion, enzymatic activity, protein–protein interactions, and folding, unfolding, and refolding [ 5 , 6 ]. Several studies have proposed the use of high concentrations of natural and synthetic macromolecules to study crowding [ 7 , 8 , 9 , 10 ], and encapsulating proteins within the pores of silica, polyacrylamide, or other hydrogels to study confinement [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. These studies have contributed to the mechanistic understanding of how proteins behave in vivo, as well as to unravel the differences between how confinement and crowding influence protein function [ 14 , 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Combined Effects of Confinement and Macromolecular Crowding on Protein Stability

Ross

Kunkel

Long

et al. 2020

IJMS

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Confinement and crowding have been shown to affect protein fates, including folding, functional stability, and their interactions with self and other proteins. Using both theoretical and experimental studies, researchers have established the independent effects of confinement or crowding, but only a few studies have explored their effects in combination; therefore, their combined impact on protein fates is still relatively unknown. Here, we investigated the combined effects of confinement and crowding on protein stability using the pores of agarose hydrogels as a confining agent and the biopolymer, dextran, as a crowding agent. The addition of dextran further stabilized the enzymes encapsulated in agarose; moreover, the observed increases in enhancements (due to the addition of dextran) exceeded the sum of the individual enhancements due to confinement and crowding. These results suggest that even though confinement and crowding may behave differently in how they influence protein fates, these conditions may be combined to provide synergistic benefits for protein stabilization. In summary, our study demonstrated the successful use of polymer-based platforms to advance our understanding of how in vivo like environments impact protein function and structure.

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Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder

Cited by 33 publications

References 125 publications

α-Lactalbumin, Amazing Calcium-Binding Protein

α-Lactalbumin, Amazing Calcium-Binding Protein

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

Combined Effects of Confinement and Macromolecular Crowding on Protein Stability

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