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Related Concept Videos

Protein Folding01:22

Protein Folding

Overview
Protein Folding01:25

Protein Folding

Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...
Protein Denaturation01:28

Protein Denaturation

The function of proteins depends on their native three-dimensional structure, which is dictated by the amino acid sequence of the specific protein. Folding of the polypeptide chain takes place under specific conditions that energetically favor the folded conformation. In contrast, protein denaturation occurs spontaneously under unfavorable conditions that disrupt the integrity of the folded conformation. Thus, the chemical and physical environment of a protein, such as significant changes in pH...
Molecular Chaperones and Protein Folding03:00

Molecular Chaperones and Protein Folding

The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...

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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
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Determining denaturation midpoints in multiprobe equilibrium protein folding experiments.

Athi N Naganathan1, Victor Muñoz

  • 1Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742, USA. vmunoz@cib.csic.es

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|June 11, 2008
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Summary

Multiprobe unfolding experiments can achieve atomic resolution. Statistical analysis reveals that derivative methods offer more accurate denaturation midpoints (Tm) than two-state fits, especially with incomplete data, improving protein folding studies.

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Area of Science:

  • Biophysics
  • Protein Folding Dynamics
  • Statistical Mechanics

Background:

  • Multiprobe equilibrium unfolding experiments can resolve protein folding at atomic resolution.
  • Unfolding curves are characterized by their denaturation midpoint (Tm), often determined using two-state models.
  • Heterogeneity in unfolding curves can arise from experimental uncertainty or true atomic-level differences.

Purpose of the Study:

  • To analyze the accuracy of Tm determination from derivative extrema versus two-state fits under various simulated conditions.
  • To develop methods for distinguishing atomic-level unfolding heterogeneity from experimental noise.
  • To assess Tm accuracy and potential biases in multiprobe NMR experiments of the BBL protein.

Main Methods:

  • Statistical analysis of simulated unfolding curves under different experimental conditions.
  • Comparison of Tm accuracy derived from derivative extrema and two-state fitting.
  • Application of developed discrimination procedures to experimental data from BBL protein multiprobe NMR studies.

Main Results:

  • Both derivative and two-state methods yield Tm accuracy better than 1.8 K for BBL, contrasting with the experimentally observed 60 K spread.
  • Incomplete baselines in unfolding curves systematically shift Tm values in two-state fits, underestimating the true spread.
  • The derivative method is more robust to incomplete baselines and broad Tm distributions, making it preferable for multiprobe experiments.

Conclusions:

  • The 60 K Tm spread in BBL is a lower limit, likely due to experimental artifacts and fitting biases.
  • Derivative-based Tm determination is superior for multiprobe unfolding experiments with heterogeneous data.
  • This work provides a foundation for rigorous quantitative analysis of future fast protein folding studies.