nmr

What are the four levels of protein structure?

Primary, secondary (helices/sheets), tertiary (3D fold), quaternary (multi-subunit complex).

Why are proteins studied using NMR?

NMR allows structural and dynamic study in solution or native-like environments.

List 3 advantages of protein NMR.

(1) Works in solution or cells, (2) sees dynamics, (3) detects weak/transient interactions.

List 3 disadvantages of protein NMR.

(1) Low sensitivity, (2) limited to ~30 kDa proteins, (3) requires costly 13C/15N labeling.

What are the steps in a typical protein NMR study?

Label protein → acquire spectra → assign resonances → build structure (optional) → analyze dynamics.

Why are 2D/3D/4D NMR spectra used in protein studies?

To resolve overlapping peaks in complex spectra.

What does a well-folded protein look like in a 1D 1H spectrum?

Wide signal dispersion across the spectrum.

What does COSY detect?

Proton-proton couplings within 3 bonds (scalar coupling).

What does TOCSY show?

All protons within a single amino acid side chain (through-bond network).

What does NOESY detect?

Spatially close protons (<5 Å), used to derive distances for structure.

What is the main data type used to build 3D protein structures in NMR?

NOE distance constraints from NOESY spectra.

What is the typical result of an NMR structure calculation?

An ensemble of similar 3D structures.

Why are 13C and 15N used in protein NMR?

They improve resolution and reduce signal overlap.

What is the purpose of a 15N-1H HSQC spectrum?

It gives a fingerprint of all backbone NH pairs in the protein.

How is isotope-labeled protein produced?

By expressing it in bacteria grown in 13C/15N-enriched minimal media.

Why use 3D or 4D NMR?

To separate overlapping signals by spreading them into additional dimensions.

How are 3D spectra visualized?

As 2D slices along one of the frequency axes.

How can secondary structure be predicted using NMR?

By comparing chemical shifts of Cα and Cβ atoms to random coil values.

What does the Ramachandran plot show?

Allowed φ and ψ backbone angles for amino acids.

What does RDC (Residual Dipolar Coupling) measure?

Orientation of bond vectors relative to the molecule's alignment axis.

What does PRE (Paramagnetic Relaxation Enhancement) measure?

Long-range distances via signal broadening from a paramagnetic probe.

Why are advanced algorithms used in structure calculation?

To handle ambiguous NOE data and assign peaks during model building.