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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...
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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.
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Allostery without Conformational Change: A Native Mass Spectrometry Perspective.

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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.

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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.