scholarly journals Room-temperature Storage of Lyophilized Engineered Bacteria using Tardigrade Intrinsically Disordered Proteins

2021 ◽  
Author(s):  
Yixian Yang ◽  
Zhandong Jiao ◽  
Shao Zhang ◽  
Mingjian Shan ◽  
Sizhe Duan ◽  
...  
Keyword(s):  
Room Temperature ◽  
Intrinsically Disordered Proteins ◽  
Survival Rates ◽  
Genetically Engineered ◽  
Disordered Proteins ◽  
High Survival ◽  
Room Temperature Storage ◽  
Intrinsically Disordered ◽  
Engineered Bacteria ◽  
Temperature Storage

Tardigrades, which live in transiently wet environments such as moss, are well-known for their extreme resistance to desiccation. Tardigrade intrinsically disordered proteins (TDPs) have been reported to also protect bacteria and yeast under desiccation. In this study, we utilized lyophilization to achieve room-temperature storage of engineered bacteria. By using TDPs, engineered bacteria are protected under lyophilization and their original functions are preserved. This study shows that TDPs can be expressed in the Escherichia coli (E. coli) BL21 and DH5α, and bacteria treated with Cytosolic-abundant heat soluble protein (CAHS) 106094 displayed the highest survival rate after lyophilization. Moreover, this study shows that the co-expression of TDPs can improve the preservation of bacteria and maintain high survival rates after prolonged room temperature storage. Additionally, the TDPs can be expressed using different vectors, which means that they can be used in different types of engineered bacteria. This study offers a new storage method that not only improves the storage of biological material for industrial and daily usage, but also for future iGEM (International Genetically Engineered Machine Competition) teams to store and use their engineered bacteria in different applications.

Sequence Specificity Despite Intrinsic Disorder: How a Disease-Associated Val/Met Polymorphism Shifts Tertiary Interactions in a Long Disordered Protein

2019 ◽  
Author(s):  
Ruchi Lohia ◽  
Reza Salari ◽  
Grace Brannigan
Keyword(s):  
Secondary Structure ◽  
Electrostatic Interactions ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Sequence Specificity ◽  
Intrinsically Disordered ◽  
Specific Effects ◽  
Heterogeneous Polymer ◽  
Non Local ◽  
Dynamics Simulations

<div>The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) encodes a hydrophobic-to-hydrophobic mutation at the midpoint of the prodomain of precursor brain-derived neurotrophic factor (BDNF), one of the earliest SNPs to be associated with neuropsychiatric disorders, for which the underlying molecular mechanism is unknown. Here we report on over 250 μs of fully-atomistic, explicit solvent, temperature replica exchange molecular dynamics simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence.</div><div>The simulations were able to correctly reproduce the location of both local and non-local secondary changes due to the Val66Met mutation when compared with NMR spectroscopy. We find that the local structure change is mediated via entropic and sequence specific effects. We show that the highly disordered prodomain can be meaningfully divided into domains based on sequence alone. Monte Carlo simulations of a self-excluding heterogeneous polymer, with monomers representing each domain, suggest the sequence would be effectively segmented by the long, highly disordered polyampholyte near the sequence midpoint. This is qualitatively consistent with observed interdomain contacts within the BDNF prodomain, although contacts between the two segments are enriched relative to the self-excluding polymer. The Val66Met mutation increases interactions across the boundary between the two segments, due in part to a specific Met-Met interaction with a Methionine in the other segment. This effect propagates to cause the non-local change in secondary structure around the second methionine, previously observed in NMR. The effect is not mediated simply via changes in inter-domain contacts but is also dependent on secondary structure formation around residue 66, indicating a mechanism for secondary structure coupling in disordered proteins. </div>

Current Challenges and Limitations in the Studies of Intrinsically Disordered Proteins in Neurodegenerative Diseases by Computer Simulations

2020 ◽  
Vol 17 ◽  
Author(s):  
Ibrahim Yagiz Akbayrak ◽  
Sule Irem Caglayan ◽  
Zilan Ozcan ◽  
Vladimir N. Uversky ◽  
Orkid Coskuner-Weber
Keyword(s):  
Neurodegenerative Diseases ◽  
Computer Simulations ◽  
Conformational Changes ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Computational Studies ◽  
Accurate Data ◽  
Aggregation Propensity ◽  
Intrinsically Disordered ◽  
Future Developments

: Experiments face challenges in the analysis of intrinsically disordered proteins in solution due to fast conformational changes and enhanced aggregation propensity. Computational studies complement experiments, being widely used in the analyses of intrinsically disordered proteins, especially those positioned at the centers of neurodegenerative diseases. However, recent investigations – including our own – revealed that computer simulations face significant challenges and limitations themselves. In this review, we introduced and discussed some of the scientific challenges and limitations of computational studies conducted on intrinsically disordered proteins. We also outlined the importance of future developments in the areas of computational chemistry and computational physics that would be needed for generating more accurate data for intrinsically disordered proteins from computer simulations. Additional theoretical strategies that can be developed are discussed herein.

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