scholarly journals Effect of protein–protein interactions and solvent viscosity on the rotational diffusion of proteins in crowded environments

2019 ◽  
Vol 21 (2) ◽  
pp. 876-883 ◽  
Author(s):  
Grzegorz Nawrocki ◽  
Alp Karaboga ◽  
Yuji Sugita ◽  
Michael Feig
Keyword(s):  
Protein Interactions ◽  
Rotational Diffusion ◽  
Protein Protein Interactions ◽  
Solvent Viscosity ◽  
Close Proximity ◽  
Crowded Environments

Slow-down of the rotational diffusion of villin in the presence of villin crowder in close proximity.

scholarly journals Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Christopher R. Horne ◽  
Hariprasad Venugopal ◽  
Santosh Panjikar ◽  
David M. Wood ◽  
Amy Henrickson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Dna Binding ◽  
Protein Interactions ◽  
Acid Metabolism ◽  
Environmental Changes ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Nutrient Source ◽  
Cryo Electron Microscopy ◽  
Close Proximity

AbstractBacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

scholarly journals In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 142 ◽  
Author(s):  
Arman Kulyyassov ◽  
Vasily Ogryzko
Keyword(s):  
Transcription Factors ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Quantitative Estimation ◽  
Multiple Reaction Monitoring ◽  
Negative Control ◽  
Protein Protein Interactions ◽  
Close Proximity ◽  
Green Fluorescent

Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.

Protein–Protein Interactions Affect Alpha Helix Stability in Crowded Environments

2015 ◽  
Vol 119 (7) ◽  
pp. 2956-2967 ◽  
Author(s):  
Bryanne Macdonald ◽  
Shannon McCarley ◽  
Sundus Noeen ◽  
Alan E. van Giessen
Keyword(s):  
Protein Interactions ◽  
Alpha Helix ◽  
Protein Protein Interactions ◽  
Helix Stability ◽  
Crowded Environments

scholarly journals Protein-Protein Interactions Affect Native State Stability in Crowded Environments

2015 ◽  
Vol 108 (2) ◽  
pp. 52a
Author(s):  
Alan E. van Giessen ◽  
Bryanne Macdonald ◽  
Shannon McCarley ◽  
Sundus Noeen ◽  
Rabeb Layouni
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Native State ◽  
Crowded Environments

scholarly journals Protein Chemical Labeling Using Biomimetic Radical Chemistry

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3980 ◽  
Author(s):  
Sato ◽  
Nakamura
Keyword(s):  
Protein Interactions ◽  
Protein Denaturation ◽  
Radical Reaction ◽  
Protein Labeling ◽  
Protein Protein Interactions ◽  
Chemical Labeling ◽  
C Terminus ◽  
Aqueous Solvent ◽  
Close Proximity ◽  
Recent Trends

Chemical labeling of proteins with synthetic low-molecular-weight probes is an important technique in chemical biology. To achieve this, it is necessary to use chemical reactions that proceed rapidly under physiological conditions (i.e., aqueous solvent, pH, low concentration, and low temperature) so that protein denaturation does not occur. The radical reaction satisfies such demands of protein labeling, and protein labeling using the biomimetic radical reaction has recently attracted attention. The biomimetic radical reaction enables selective labeling of the C-terminus, tyrosine, and tryptophan, which is difficult to achieve with conventional electrophilic protein labeling. In addition, as the radical reaction proceeds selectively in close proximity to the catalyst, it can be applied to the analysis of protein–protein interactions. In this review, recent trends in protein labeling using biomimetic radical reactions are discussed.

scholarly journals Quantifying Protein-Protein Interactions in Molecular Simulations

2019 ◽  
Author(s):  
Alfredo Jost Lopez ◽  
Patrick K. Quoika ◽  
Max Linke ◽  
Gerhard Hummer ◽  
Juergen Koefinger
Keyword(s):  
Molecular Dynamics ◽  
Protein Interactions ◽  
Virial Coefficient ◽  
Analytical Ultracentrifugation ◽  
Molecular Simulations ◽  
Simple Expression ◽  
Protein Protein Interactions ◽  
Explicit Solvent ◽  
Dynamics Simulations ◽  
Crowded Environments

<p><a></a></p><p><a></a>We present simple, accurate, and efficient methods to estimate the dissociation constant K<sub>d </sub>and the second osmotic virial coefficient B<sub>2 </sub>from molecular simulations. We show that for simulations of two proteins in a box, K<sub>d </sub>is determined by B<sub>2 </sub>and the fraction of bound protein. We present two different methods to calculate B<sub>2 </sub>from Monte Carlo and molecular dynamics simulations using implicit or explicit solvent. We derive a surprisingly simple expression for B<sub>2</sub>, adding significantly to the understanding of this important quantity. Non-binding interactions of proteins and other macromolecules shape the physicochemical properties of the crowded environments inside cells and of biomolecular condensates. We show how to extract the contributions of non-binding conformations to B<sub>2 </sub>and discuss how these can be determined in analytical ultracentrifugation and SAXS experiments. We expect that our methods will prove to be instrumental in force parameterization efforts and high-throughput studies of large interactomes. </p>

scholarly journals Quantifying Protein-Protein Interactions in Molecular Simulations

2019 ◽  
Author(s):  
Alfredo Jost Lopez ◽  
Patrick K. Quoika ◽  
Max Linke ◽  
Gerhard Hummer ◽  
Juergen Koefinger
Keyword(s):  
Molecular Dynamics ◽  
Protein Interactions ◽  
Virial Coefficient ◽  
Analytical Ultracentrifugation ◽  
Molecular Simulations ◽  
Simple Expression ◽  
Protein Protein Interactions ◽  
Explicit Solvent ◽  
Dynamics Simulations ◽  
Crowded Environments

<p><a></a></p><p><a></a>We present simple, accurate, and efficient methods to estimate the dissociation constant K<sub>d </sub>and the second osmotic virial coefficient B<sub>2 </sub>from molecular simulations. We show that for simulations of two proteins in a box, K<sub>d </sub>is determined by B<sub>2 </sub>and the fraction of bound protein. We present two different methods to calculate B<sub>2 </sub>from Monte Carlo and molecular dynamics simulations using implicit or explicit solvent. We derive a surprisingly simple expression for B<sub>2</sub>, adding significantly to the understanding of this important quantity. Non-binding interactions of proteins and other macromolecules shape the physicochemical properties of the crowded environments inside cells and of biomolecular condensates. We show how to extract the contributions of non-binding conformations to B<sub>2 </sub>and discuss how these can be determined in analytical ultracentrifugation and SAXS experiments. We expect that our methods will prove to be instrumental in force parameterization efforts and high-throughput studies of large interactomes. </p>

Cross-saturation and transferred cross-saturation experiments

2014 ◽  
Vol 47 (2) ◽  
pp. 143-187 ◽  
Author(s):  
Takumi Ueda ◽  
Koh Takeuchi ◽  
Noritaka Nishida ◽  
Pavlos Stampoulis ◽  
Yutaka Kofuku ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Protein Protein Interactions ◽  
Molecular Weights ◽  
Protein Ligands ◽  
Close Proximity ◽  
Complex Models ◽  
Functional Mechanisms

AbstractStructural analyses of protein–protein interactions are required to reveal their functional mechanisms, and accurate protein–protein complex models, based on experimental results, are the starting points for drug development. In addition, structural information about proteins under physiologically relevant conditions is crucially important for understanding biological events. However, for proteins such as those embedded in lipid bilayers and transiently complexed with their effectors under physiological conditions, structural analyses by conventional methods are generally difficult, due to their large molecular weights and inhomogeneity. We have developed the cross-saturation (CS) method, which is an nuclear magnetic resonance measurement technique for the precise identification of the interfaces of protein–protein complexes. In addition, we have developed an extended version of the CS method, termed transferred cross-saturation (TCS), which enables the identification of the residues of protein ligands in close proximity to huge (>150 kDa) and heterogeneous complexes under fast exchange conditions (>0.1 s−1). Here, we discuss the outline, basic theory, and practical considerations of the CS and TCS methods. In addition, we will review the recent progress in the construction of models of protein–protein complexes, based on CS and TCS experiments, and applications of TCS to in situ analyses of biologically and medically important proteins in physiologically relevant states.

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