Dataset Information

Action-FRET of a Gaseous Protein.

ABSTRACT: Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question-and thus further develop mass spectrometry as a tool for structural biology-structure-sensitive techniques must be developed to probe the gas-phase conformations of proteins. Here, we report Förster resonance energy transfer (FRET) measurements on a ubiquitin mutant using specific photofragmentation as a reporter of the FRET efficiency. The FRET data is interpreted in the context of circular dichroism, molecular dynamics simulation, and ion mobility data. Both the dependence of the FRET efficiency on the charge state-where a systematic decrease is observed-and on methanol concentration are considered. In the latter case, a decrease in FRET efficiency with methanol concentration is taken as evidence that the conformational ensemble of gaseous protein cations retains a memory of the solution phase conformational ensemble upon electrospray ionization. Graphical Abstract ?.

SUBMITTER: Daly S

PROVIDER: S-EPMC5174150 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Action-FRET of a Gaseous Protein.

Daly Steven S Knight Geoffrey G Halim Mohamed Abdul MA Kulesza Alexander A Choi Chang Min CM Chirot Fabien F MacAleese Luke L Antoine Rodolphe R Dugourd Philippe P

Journal of the American Society for Mass Spectrometry 20160809 1

Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question-and thus further develop mass spectrometry as a tool for structural biology-structure-sensitive techniques must be developed to probe the gas-phase conformations of prote ...[more]

PMID: 27506208

Similar Datasets

Project description:Our intention is to provide easy methods for estimating entropy and chemical potentials for gas phase reactions. Clausius' virial theorem set a basis for relating kinetic energy in a body of independent material particles to its potential energy, pointing to their complementary role with respect to the second law of maximum entropy. Based on this partitioning of thermal energy as sensible heat and also as a latent heat or field potential energy, in action mechanics we express the entropy of ideal gases as a capacity factor for enthalpy plus the configurational work to sustain the relative translational, rotational, and vibrational action. This yields algorithms for estimating chemical reaction rates and positions of equilibrium. All properties of state including entropy, work potential as Helmholtz and Gibbs energies, and activated transition state reaction rates can be estimated, using easily accessible molecular properties, such as atomic weights, bond lengths, moments of inertia, and vibrational frequencies. We conclude that the large molecular size of many enzymes may catalyze reaction rates because of their large radial inertia as colloidal particles, maximising action states by impulsive collisions. Understanding how Clausius' virial theorem justifies partitioning between thermal and statistical properties of entropy, yielding a more complete view of the second law's evolutionary nature and the principle of maximum entropy. The ease of performing these operations is illustrated with three important chemical gas phase reactions: the reversible dissociation of hydrogen molecules, lysis of water to hydrogen and oxygen, and the reversible formation of ammonia from nitrogen and hydrogen. Employing the ergal also introduced by Clausius to define the reversible internal work overcoming molecular interactions plus the configurational work of change in Gibbs energy, often neglected; this may provide a practical guide for managing industrial processes and risk in climate change at the global scale. The concepts developed should also have value as novel methods for the instruction of senior students.

Project description:The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.

Dataset Information

Action-FRET of a Gaseous Protein.

Publications

Action-FRET of a Gaseous Protein.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets