Question

In NMR, what kind of information do you get when you collect a 15N-1H HSQC spectrum of a protein?

a. Interactions between protons < 5Å apart.

b.A bunch of peaks

c.The 2D spectrum of all the amide N's and their attached protons.

d. A 3D spectrum

Answer 1

In ¹⁵N-¹H HSQC spectrum of a protein, we can measure short proton-proton distances and angles. We can make out secondary and tertiary structure of proteins. Folded and unfolded proteins can be characterised.

a. Interactions between protons < 5Å apart:

The Nuclear Overhauser Effect is the change (enhancement) of the signal intensity from a given nucleus as a result of exciting or saturating the resonance frequency of another nucleus. It operates through-space interactions and the magnitude of the effect is dependent on distance -enhancement depends on 1/r6, where r is the internuclear distance -thus, the effect is limited to distances of approximately 5Å or less. This provides a means to determine if any two protons in a protein are < 5Å apart.

b. A bunch of peaks :

For even small proteins, 1D spectra are complicated and cannot be analyzed comprehensively but can be useful for evaluating the suitability/stability.

For properly folded small proteins, peaks should be sharp and should show good chemical shift dispersion (i.e. tertiary structure intact). For unfolded proteins, peaks are usually broad (many protons in each peak) and chemical shift dispersion is poor (leading to the broad peaks). Backbone NH (8.1-8.5 ppm) in water and CH₃ (0.8-2ppm) chemical shifts are characterised in unfolded protein. A folded protein generally gives NMR signals of 8.7 ppm or larger for backbone NH in water, and signals of less than 0.5 ppm for CH₃.

c. The 2D spectrum of all the amide N's and their attached protons:

In the 2D spectrum at right, each backbone amide gives rise to a cross peak at the coordinate given by the 1HNand 15N frequencies. Can be used to display characterization of protein foldness.

d. A 3D spectrum:

Provide intra- and inter- residue connectivity and correlates Cα and Cβ to the backbone -NH group within the same amino acid. Provide inter- residue connectivity and correlates Cα and Cβ to the backbone NH group. Combining information, one can connect the amino acid by walking along the polypeptide chain.