Allostery without Conformational Change: A Native Mass Spectrometry Perspective.
He Mirabel Sun1, Kacie A Evans1, Morgan Powers2
1Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
The Journal of Physical Chemistry. B
|August 19, 2025
Summary
Variable temperature native electrospray ionization-mass spectrometry (vT-nESI-MS) reveals how buffer choice impacts nucleotide binding in the GroEL single ring mutant (SR1). Temperature and buffer conditions significantly alter protein dynamics and binding thermodynamics, suggesting allostery without major structural changes.
Area of Science:
- Biochemistry and Biophysics
- Mass Spectrometry
- Protein Dynamics
Background:
- Native electrospray ionization-mass spectrometry (nESI-MS) is crucial for studying intact proteins and their complexes.
- Variable temperature (vT)-nESI-MS enhances thermodynamic studies of protein-ligand interactions.
- The GroEL single ring mutant (SR1) serves as a model system for investigating protein-nucleotide binding.
Purpose of the Study:
- To investigate the effects of temperature and native ESI buffers on nucleotide (ADP) binding to SR1.
- To determine ADP binding affinities, cooperativity, and enthalpy-entropy compensation (EEC) under varying conditions.
- To elucidate how buffer composition influences SR1 conformation, stability, and dynamics.
Main Methods:
- Utilized variable temperature native electrospray ionization-mass spectrometry (vT-nESI-MS).
- Employed ion mobility spectrometry (IMS) to analyze collision cross sections (CCS).
- Compared three common native ESI buffers: ammonium acetate (AmAc), triethylammonium acetate (TEAA), and ethylenediammonium acetate (EDDA).
Main Results:
- Temperature-dependent shifts in average charge states (Z_avg) and CCS indicated buffer-induced alterations in SR1 structure, stability, and dynamics.
- ADP binding cooperativity varied significantly with temperature and buffer type: highly cooperative in AmAc at low temperatures, less so in TEAA, and absent in EDDA.
- Observed enthalpy-entropy compensation (EEC) and changes in heat capacity suggest ADP binding influences SR1 conformational states, despite no large-scale structural changes detected by IMS.
Conclusions:
- Buffer composition and temperature critically modulate SR1 dynamics and nucleotide binding thermodynamics.
- The findings support the concept of 'allostery without (measurable) conformational change' for SR1-ADP binding.
- Results align with studies on hydration effects in protein folding and ligand binding, highlighting the role of solvent interactions.
More Related Videos
07:33Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry
Published on: October 15, 2018
14.4K
09:34Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry
Published on: September 20, 2016
11.3K
Related Concept Videos
01:19Mass Spectrometry: Overview
5.8K
Mass spectrometry is an analytical technique used to determine the molecular mass and molecular formula of a compound. The basic principle of mass spectrometry is to generate ions from the analyte molecule and measure these ion abundances against their molecular mass. One common type of ionization, known as electrospray ionization or EI, bombards the analyte molecules in the gas phase with high-energy electron beams. The electron beams displace an electron from the molecule and leave...
5.8K
01:24Mass Spectrum: Interpretation
1.6K
An unknown compound can be established by identifying the molecular ion peak in the mass spectrum. The molecular ion peak is often weak or absent due to the predominance of fragmentation in high-energy electron beams. In such cases, a low-energy electron beam can be used to scan the spectrum to enhance the intensity of the molecular ion peak. Additionally, chemical ionization, field ionization, and desorption ionization spectra are used to obtain a relatively intense molecular ion peak.
To...
To...
1.6K
01:13Mass Spectrometry: Isotope Effect
2.5K
Most elements exist in nature as a mixture of isotopes. The isotopes differ in weight due to their respective number of neutrons. The molecular weight of a molecule is different depending on the specific isotope of its elements involved. As a result, the mass spectrum of the molecule exhibits peaks from the same fragment at multiple positions. The positions of these mass signals depend on the difference between the molecular mass. Furthermore, the intensity of these signals is dependent on the...
2.5K
01:15High-Resolution Mass Spectrometry (HRMS)
1.6K
The resolution of a mass spectrometer depends on the efficiency of separating ions with different ion masses. The mass of an atom is approximated to the sum of the masses of protons and neutrons inside, considering the masses of protons and neutrons as equal. However, the masses of the proton (1.6726 × 10−24 g) and neutron (1.6749 × 10−24 g) are not truly equal. There is a minor error in the expression of atomic masses relative to the simplest atom of hydrogen. For...
1.6K
01:21Mass Spectrometry: Complex Analysis
889
Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
889
00:53Mass Spectrometry: Alkyne Fragmentation
1.6K
The fragmentation of alkynes preferentially occurs at the carbon–carbon bond between the α and β carbon of the alkyne bond to generate a 3-propynyl cation (or propargyl cation). In terminal alkynes, there is the only type of fragmentation that yields the 3-propynyl cation. The unsubstituted 3-propynyl cation exhibits a peak at a mass-to-charge ratio of 39. In internal alkynes, the 3-propynyl cation is substituted. For example, 2-pentyne fragments into methyl-substituted...
1.6K