Mixed Macromolecular Crowding: A Protein and Solvent Perspective

Biswas, Saikat; Kundu, Jayanta; Mukherjee, Sumitava; Chowdhury, P. K.

doi:10.1021/acsomega.7b01864

Cited by 48 publications

(44 citation statements)

References 92 publications

(190 reference statements)

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“…Under these conditions, rate enhancement of hydride transfer from excluded volume effects is counteracted by slowed diffusion and impeded product release. These results support the claim of a previous study that the smaller crowder in a binary mixture dictates the overall effects of that mixture [ 60 ], because the relative V max values of the mixtures were more similar to individual solutions of 10 and 40 kDa dextran, rather than 500 kDa dextran. Unlike previous work [ 17 , [53] , [54] , [55] , [56] , [57] ], our results suggest that effects from mixtures are comparable to the effects of the individual crowders.…”

Section: Discussionsupporting

confidence: 90%

“…One study using fluorescence correlation spectroscopy (FCS) to monitor small molecule diffusion found that the microviscosity of binary crowding mixtures was greater than the sum of the microviscosities for the individual crowding agents. Since deviations between mixture effects and the sum of its parts was greatest for the largest crowders (70 kDa dextran and 70 kDa Ficoll), the authors attributed the increased microviscosity to crowder entanglement [ 60 ]. Finally, this same work showed that binary mixtures containing 6 kDa dextran had the similar effects on the domain movements of a protein regardless of the identity of the other dextran (40 or 70 kDa) or Ficoll (70 kDa), suggesting that the smaller component in a binary mixture dictates the overall effects.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 90%

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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“…PEG has been used in many studies on crowding [ 17 , 19 ]. PEG8000 (8 kDa), used in this study, is not an ideal crowding agent, as it is known to interact with proteins at higher concentrations [ 28 ]. In dilute conditions, PEG8000 attains a globular shape with a neutrally charged surface [ 29 ].…”

Section: Introductionmentioning

confidence: 99%

Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU

Lin

Wuite

Dame

2020

IJMS

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HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

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“…4), possibly acting as an agent of MC, decreasing entropic forces and leading to segregation ( 80 ). For example, a study shows that certain molecular crowders (dextran, Ficoll, PEG, and human serum albumin) that have chemical similarities with capsicumicine (hydrophilicity, linear open chains, and neutrality) modulate refolding kinetics and decrease competing aggregation and segregation ( 81 ).…”

Section: Discussionmentioning

confidence: 99%

First-in-class matrix anti-assembly peptide prevents staphylococcal biofilm in vitro and in vivo

Borowski

Chat

Schneider

et al. 2020

Preprint

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15 Biofilm-forming bacteria are an important reservoir of nosocomial infections, and source of 16 antibiotic resistance and tolerance. These bacteria are enclosed in a complex matrix that allows 17 them to adhere to medical devices and tissue, and which also protects against antibiotics and 18 immune systems. Advances in antibiofilm strategies for targeting the biofilm matrix are therefore 19 extremely relevant in the fight against multidrug-resistant and/or -tolerant bacteria. Plants are 20 constantly attacked by a wide range of pathogens, and have protective factors such as peptides to 21 defend themselves. These peptides are common components in Capsicum red pepper seeds, and 22here we investigate and identify a new antibiofilm peptide, "capsicumicine." We demonstrate that 23 capsicumicine prevents methicillin-resistant Staphylococcus epidermidis adhesion as well as biofilm 24 establishment and maintenance via a new extracellular "matrix anti-assembly" (MAA) mechanism 25 of action. In this manner, capsicumicine disturbs the construction of the matrix, and therefore its 26 functioning. Importantly, the peptide is not an antibiotic and it is not cytotoxic, thus capsicumicine 27 is an exciting new alternative treatment for prevention of biofilm infections. 28Significance 30 Both pathogenic drug resistance and tolerance are favored by biofilm development. Indeed, biofilm-31 covered microorganisms display antibiotic resistance up to 1000 times higher than planktonic ones. 32Staphylococcus epidermidis is remarkably good at forming biofilm, and is therefore highly associated 33 with medical device infection. Here, we report the discovery of "capsicumicine," a novel antibiofilm 34 peptide derived from Capsicum bacattum. This prevents the establishment of S. epidermidis biofilm 35 via a new mechanism of action, matrix anti-assembly (MAA). MAA is not antibiotic, and is based on 36 extracellular interactions that force bacteria to remain planktonic, letting immune systems and 37 2 traditional antibiotics more easily clear the infection. For this reason, MAA should confer a lower 38 susceptibility to bacterial resistance development. Very importantly, capsicumicine is not cytotoxic. 39 Introduction 40Antimicrobial failure is a worldwide challenge, currently addressed by a WHO global action plan (1). A 41 lack of new antibiotics and the inappropriate use of older treatments mean that multidrug-resistant 42 strains are increasing (2). This process is favored by biofilm development, and microorganisms 43 enclosed in biofilm matrix have antibiotic resistance that is up to 1000 times higher than planktonic 44 ones (3-5). This makes the matrix itself an important target for biofilm control. Biofilms are organized 45 microbial clusters made of a self-assembled matrix that usually attaches to a surface, whether abiotic 46 (medical devices, teeth, etc.) or biotic (host tissues, mucus, inside chronic wounds, etc.) (6, 7). Since 47 the bacteria are embedded into this self-assembled matrix, they are harder to treat because of their 4...

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Mixed Macromolecular Crowding: A Protein and Solvent Perspective

Cited by 48 publications

References 92 publications

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

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

Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU

First-in-class matrix anti-assembly peptide prevents staphylococcal biofilm in vitro and in vivo

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