Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL

Pastor, Ashutosh; Singh, Amit Kumar; Fisher, Mark T.; Chaudhuri, Tapan K.

doi:10.1111/febs.13796

Cited by 5 publications

(7 citation statements)

References 27 publications

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“…This would be consistent with the fact that the majority of GroES release was shown to occur via polarity change (69%) by way of a football complex (Yamamoto and Ando, 2016). Another reason for retaining such a cooperative system could be the fact that GroEL is able to fold large proteins that cannot be accommodated inside the cavity underneath the GroES (Chaudhuri et al, 2009; Dahiya and Chaudhuri, 2014; Pastor et al, 2016). In this instance, it is possible that release of the cis -bound substrate must be induced by trans binding of substrate, ATP and GroES in a fully alternating mechanism, although in this case, the folding protein would not have the benefit of encapsulation.…”

Section: Recent Developments and Outstanding Questionsmentioning

confidence: 99%

Dynamic Complexes in the Chaperonin-Mediated Protein Folding Cycle

Weiss

Jebara

Nisemblat

et al. 2016

Front. Mol. Biosci.

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The GroEL–GroES chaperonin system is probably one of the most studied chaperone systems at the level of the molecular mechanism. Since the first reports of a bacterial gene involved in phage morphogenesis in 1972, these proteins have stimulated intensive research for over 40 years. During this time, detailed structural and functional studies have yielded constantly evolving concepts of the chaperonin mechanism of action. Despite of almost three decades of research on this oligomeric protein, certain aspects of its function remain controversial. In this review, we highlight one central aspect of its function, namely, the active intermediates of its reaction cycle, and present how research to this day continues to change our understanding of chaperonin-mediated protein folding.

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Section: Recent Developments and Outstanding Questionsmentioning

confidence: 99%

Dynamic Complexes in the Chaperonin-Mediated Protein Folding Cycle

Weiss

Jebara

Nisemblat

et al. 2016

Front. Mol. Biosci.

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“…To optimize the refolding of mutant R52Tat without EGFP, we compared the effect of an uncrowded environment on the TatR52A (purified) in E. coli cells with that of a crowded environment on EGFP in the absence of TAR RNA by BLI at 1.3 nm during a refolding period of 20 min ( Figures S4 and S5 ) [ 56 ]. We used this well-characterized system to assess the response of His-tagged Tat-BLI after binding to the TAR RNA substrate to design a BLI-detection study for mutant R52Tat folding without EGFP in the absence of TAR RNA ( Figure 5 and Figure S4 ) [ 3 , 57 ].…”

Section: Resultsmentioning

confidence: 99%

“…The fold changes for each mutant compared with the WT values were calculated. The BLI experiments were performed using the general BLI analysis protocol, as described previously [ 56 ]. Data analysis, including double reference subtraction, was performed with the ForteBio data analysis software.…”

Section: Methodsmentioning

confidence: 99%

TAR RNA Mediated Folding of a Single-Arginine-Mutant HIV-1 Tat Protein within HeLa Cells Experiencing Intracellular Crowding

Kim

Chun

2021

IJMS

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The various effects of native protein folding on the stability and folding rate of intrinsically disordered proteins (IDPs) in crowded intracellular environments are important in biomedicine. Although most studies on protein folding have been conducted in vitro, providing valuable insights, studies on protein folding in crowded intracellular environments are scarce. This study aimed to explore the effects of intracellular molecular crowding on the folding of mutant transactivator HIV-1 Tat based on intracellular interactions, including TAR RNA, as proof of the previously reported chaperna-RNA concept. Considering that the Tat–TAR RNA motif binds RNA, we assessed the po tential function of TAR RNA as a chaperna for the refolding of R52Tat, a mutant in which the argi nine (R) residues at R52 have been replaced with alanine (A) by site-directed mutagenesis. We mon itored Tat-EGFP and Tat folding in HeLa cells via time-lapse fluorescence microscopy and biolayer interferometry using EGFP fusion as an indicator for folding status. These results show that the refolding of R52A Tat was stimulated well at a 0.3 μM TAR RNA concentration; wild-type Tat refolding was essentially abolished because of a reduction in the affinity for TAR RNA at that con centration. The folding and refolding of R52Tat were mainly promoted upon stimulation with TAR RNA. Our findings provide novel insights into the therapeutic potential of chaperna-mediated fold ing through the examination of as-yet-unexplored RNA-mediated protein folding as well as viral genetic variants that modulate viral evolutionary linkages for viral diseases inside a crowded intra cellular environment.

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“…Separate control experiments indicated the immobilized MalZ, which was completely denatured at high urea concentrations at 25 °C, refolds very slowly (t 1/2 at 25 °C of ∼10 min) relative to the wash times that were used to remove an adhering denaturant or a ligand solution. 36 It was also shown that the refolded MalZ immobilized on and released from Ni-NTA surfaces was functionally active with yields that were substantially improved compared to those of solution refolding. 36 These control measurements indicated that no substantial refolding was detected (i.e., chaperonin binding amplitudes remain high) if two separate MalZ attached tips were compared before and after a 10 s wash period.…”

Section: ■ Resultsmentioning

confidence: 99%

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

et al. 2016

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Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins.

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Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL

Cited by 5 publications

References 27 publications

Dynamic Complexes in the Chaperonin-Mediated Protein Folding Cycle

Dynamic Complexes in the Chaperonin-Mediated Protein Folding Cycle

TAR RNA Mediated Folding of a Single-Arginine-Mutant HIV-1 Tat Protein within HeLa Cells Experiencing Intracellular Crowding

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

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