scholarly journals Quantitative Analyses of the Yeast Oxidative Protein Folding Pathway In Vitro and In Vivo

2019 ◽  
Vol 31 (4) ◽  
pp. 261-274 ◽  
Author(s):  
Dave M. Beal ◽  
Emma L. Bastow ◽  
Gemma L. Staniforth ◽  
Tobias von der Haar ◽  
Robert B. Freedman ◽  
...  
Keyword(s):  
Protein Folding ◽  
Folding Pathway ◽  
Oxidative Protein Folding ◽  
Quantitative Analyses

scholarly journals GroEL/ES buffers entropic traps in folding pathway during evolution of a model substrate

2020 ◽  
Author(s):  
Anwar Sadat ◽  
Satyam Tiwari ◽  
Kanika Verma ◽  
Arjun Ray ◽  
Mudassar Ali ◽  
...  
Keyword(s):  
Protein Folding ◽  
Molecular Chaperones ◽  
Folding Pathway ◽  
Substrate Protein ◽  
Model Protein ◽  
Physical Barriers ◽  
Evolutionary Step ◽  
Entropic Traps

ABSTRACTThe folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Synthesis runs counter to directional folding of a nascent protein domain

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiuqi Chen ◽  
Nandakumar Rajasekaran ◽  
Kaixian Liu ◽  
Christian M. Kaiser
Keyword(s):  
Single Molecule ◽  
Molten Globule ◽  
Elongation Factor ◽  
Protein Domain ◽  
Folding Pathway ◽  
Native Structure ◽  
Folding Intermediates ◽  
C Terminus

Abstract Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

scholarly journals Synthesis runs counter to directional folding of a nascent protein domain

2020 ◽  
Author(s):  
Xiuqi Chen ◽  
Nandakumar Rajasekaran ◽  
Kaixian Liu ◽  
Christian M. Kaiser
Keyword(s):  
Single Molecule ◽  
Molten Globule ◽  
Elongation Factor ◽  
Protein Domain ◽  
Folding Pathway ◽  
Native Structure ◽  
Folding Intermediates ◽  
C Terminus

AbstractFolding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

Globular Protein Folding In Vitro and In Vivo

2016 ◽  
Vol 45 (1) ◽  
pp. 233-251 ◽  
Author(s):  
Martin Gruebele ◽  
Kapil Dave ◽  
Shahar Sukenik
Keyword(s):  
Protein Folding ◽  
Globular Protein

scholarly journals Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

2006 ◽  
Vol 282 (7) ◽  
pp. 4437-4446 ◽  
Author(s):  
Markus Liebscher ◽  
Günther Jahreis ◽  
Christian Lücke ◽  
Susanne Grabley ◽  
Satish Raina ◽  
...  
Keyword(s):  
Protein Folding ◽  
Minimal Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Secondary Amide ◽  
Fatty Acyl Chain ◽  
Isomerase Activity

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. Nα-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 ± 20 μg/ml. The compounds were shown to compete with fluorophore-labeled σ32-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.

In vitro and in vivo assays to assess the functions of calnexin and calreticulin in ER protein folding and quality control

Methods ◽  
2005 ◽  
Vol 35 (4) ◽  
pp. 338-347 ◽  
Author(s):  
Marie-Eve Paquet ◽  
Michael R. Leach ◽  
David B. Williams
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
In Vivo Assays

scholarly journals Redox-Active Cyclic Bis(cysteinyl)peptides as Catalysts for In Vitro Oxidative Protein Folding

2002 ◽  
Vol 9 (6) ◽  
pp. 731-740 ◽  
Author(s):  
Chiara Cabrele ◽  
Stella Fiori ◽  
Stefano Pegoraro ◽  
Luis Moroder
Keyword(s):  
Protein Folding ◽  
Oxidative Protein Folding ◽  
Redox Active

Small molecule diselenide additives for in vitro oxidative protein folding

2016 ◽  
Vol 52 (16) ◽  
pp. 3336-3339 ◽  
Author(s):  
Post Sai Reddy ◽  
Norman Metanis
Keyword(s):  
Protein Folding ◽  
Small Molecule ◽  
Oxidative Folding ◽  
Oxidative Protein Folding

Small molecule diselenides were prepared and found to enhance thein vitrooxidative folding of disulfide-rich protein.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

2020 ◽  
Author(s):  
Reem Mousa ◽  
Taghreed Hidmi ◽  
Sergei Pomyalov ◽  
Shifra Lansky ◽  
Lareen Khouri ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Crystal Structure Analysis ◽  
Folding Rate ◽  
Oxidative Folding ◽  
Functional Studies ◽  
Oxidative Protein Folding ◽  
And Function

<p>The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of <i>in vitro</i> oxidative folding of many proteins.</p><p>In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.</p><p>A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. </p>Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

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