!!! Biomolecular NMR Spectroscopy & Kinetic Assays

What magnetic field property does NMR measure in a protein sample?

NMR measures nuclear spin state transitions between alpha and beta spin states in an applied magnetic field

What spectrometer magnetic strength was used in the laboratory experiment?

A 700 MHz NMR spectrometer

Which nitrogen isotope is NMR-active and used for protein labeling?

15N is NMR-active, whereas the naturally abundant 14N isotope is NMR-inactive

Which carbon isotope is NMR-active and what is its natural abundance?

13C is the only NMR-active carbon isotope, with a natural abundance of approximately 1.1%

What does a 1D 1H NMR experiment measure in a protein sample?

It measures all hydrogen protons present in the sample simultaneously

What specific relationship is measured in a 2D 1H-15N HSQC spectrum?

Heteronuclear Single Quantum Coherence measures protons directly attached to 15N nitrogen nuclei, giving one peak per backbone amide group

Why is Proline invisible in a 2D 1H-15N HSQC spectrum?

Proline contains a cyclic imine structure that lacks a backbone amide proton (NH)

What is the purpose of the Deuterium Lock step in NMR setup?

It compensates for magnetic field drift over time by continuously monitoring and adjusting to the deuterium resonance frequency

Why is 5% to 10% D2O added to the sample solvent instead of 100% D2O?

100% D2O would cause exchangeable backbone amide protons (NH) to exchange into ND, making them invisible in 1H NMR

What is the goal of Tuning and Matching in NMR probe preparation?

It adjusts the electrical circuit impedance to approximately 50 ohms for each frequency channel to prevent radio frequency signal loss

What is the primary purpose of Shimming the magnetic field?

It adjusts auxiliary gradient coils to create a highly homogeneous magnetic field across the entire sample volume

What is P1 in NMR pulse calibration?

P1 is the length of time required for a radio frequency pulse to tip the nuclear magnetization by exactly 90 degrees into the transverse xy plane

How is the 90 degree pulse length P1 calculated experimentally?

By determining the pulse duration required for a 360 degree rotation (31.7 microseconds) and dividing that time value by 4 to get 7.92 microseconds

Why is solvent water suppression required in biological NMR experiments?

The massive H2O proton signal dwarfs the small protein signals, requiring frequency offset suppression to observe protein peaks

What reference compound is used to calibrate 0 ppm chemical shift in NMR spectra?

DSS, which contains three equivalent methyl groups that define 0 ppm by definition

Why would unassisted GTP hydrolysis yield non-linear reaction kinetics?

Without recycling, GTP concentration depletes continuously over time, causing an exponential decay in hydrolysis rate

How is a constant GTP concentration maintained during kinetic NMR assays?

Pyruvate Kinase and Phosphoenolpyruvate (PEP) are added to continuously regenerate GTP from generated GDP

What is the exact stoichiometry of the GTP recycling system?

Hydrolysis of 1 GTP molecule produces 1 GDP, which Pyruvate Kinase converts back to 1 GTP by consuming 1 PEP and generating 1 Pyruvate

How do researchers indirectly measure the rate of RAS GTP hydrolysis during the recycling assay?

By monitoring the rate of PEP peak decay and Pyruvate peak growth in sequential NMR spectra

Why must Pyruvate Kinase act faster than RAS GTPase in the kinetic assay?

To ensure GDP is converted back to GTP instantaneously so that GTP concentration never becomes rate-limiting

What structural information is derived from NMR amide peak shifts upon nucleotide exchange?

Amide NH peaks that shift correspond to residues near the nucleotide binding site, while unshifted peaks belong to distant residues

How do NMR signal intensities and peak decay rates report on local protein dynamics?

Strong sharp peaks indicate highly flexible unstructured protein regions, whereas broader peaks correspond to rigid regions