scholarly journals Supplementary material to "Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR"

2020 ◽  
Author(s):  
Rita Puglisi ◽  
Annalisa Pastore ◽  
Piero Andrea Temussi
Keyword(s):  
2D Nmr ◽  
Site Specific ◽  
Supplementary Material ◽  
Fold Stability

scholarly journals Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

2021 ◽  
Author(s):  
Rita Puglisi ◽  
Gogulan Karunanithy ◽  
D. Flemming Hansen ◽  
Annalisa Pastore ◽  
Piero Andrea Temussi
Keyword(s):  
Protein Unfolding ◽  
2D Nmr ◽  
Heat Denaturation ◽  
Specific Information ◽  
Hydrophobic Core ◽  
2D Nmr Spectroscopy ◽  
Site Specific ◽  
The Individual ◽  
Low Ionic Strength ◽  
Fold Stability

AbstractMost techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

scholarly journals The anatomy of unfolding of Yfh1 is revealed by site-specific fold stability analysis measured by 2D NMR spectroscopy

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Rita Puglisi ◽  
Gogulan Karunanithy ◽  
D. Flemming Hansen ◽  
Annalisa Pastore ◽  
Piero Andrea Temussi
Keyword(s):  
Nmr Spectroscopy ◽  
Protein Unfolding ◽  
2D Nmr ◽  
Heat Denaturation ◽  
Specific Information ◽  
Hydrophobic Core ◽  
2D Nmr Spectroscopy ◽  
Site Specific ◽  
The Individual ◽  
Fold Stability

AbstractMost techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

scholarly journals Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

2020 ◽  
Author(s):  
Rita Puglisi ◽  
Annalisa Pastore ◽  
Piero Andrea Temussi
Keyword(s):  
Protein Unfolding ◽  
2D Nmr ◽  
Heat Denaturation ◽  
Specific Information ◽  
Hydrophobic Core ◽  
2D Nmr Spectroscopy ◽  
Site Specific ◽  
The Individual ◽  
Low Ionic Strength ◽  
Fold Stability

Abstract. Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether a detailed analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

2D NMR studies on muscle and cerebral metabolism in vivo

1994 ◽  
Vol 91 ◽  
pp. 697-703 ◽  
Author(s):  
B Gillet ◽  
BT Doan ◽  
C Verre-Sebrie ◽  
O Fedeli ◽  
JC Beloeil ◽  
...  
Keyword(s):  
Cerebral Metabolism ◽  
2D Nmr ◽  
Nmr Studies
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