Protein Structure and Solution NMR Spectroscopy

Flashcards covering multidomain proteins, solution-based biophysical techniques (DSF, CD, SAXS), and biomolecular NMR spectroscopy concepts including chemical shifts, backbone assignment, and molecular dynamics.

Multidomain proteins

Proteins composed of multiple modular domains that can fold independently and are often connected by flexible linkers.

Multivalent

The property of a protein that allows it to bind to another biomolecule through multiple distinct sites.

Avidity

The increased strength of an interaction resulting from multiple binding sites between two molecules.

KIX domain

A small domain of the p300/CBP protein that can bind different peptide motifs, such as pKID, using two different binding surfaces.

Differential scanning fluorimetry (DSF)

A technique that measures tryptophan fluorescence at $\lambda_{max} = 330\,nm$ and $\lambda_{max} = 350\,nm$ as a protein is heated to determine its melting temperature ($T_{m}$).

Circular dichroism (CD)

A spectroscopic technique that uses circularly polarised light to determine the percentage of secondary structure types in a protein and monitor structural changes.

Small angle x-ray scattering (SAXS)

A solution-based technique that provides low-resolution structural information identifying global shapes, such as globular or rod-like, and monitors global structural changes.

Isotopic enrichment

The process of labeling protein samples with specifically detectable nuclei, typically $^{13}C$ and/or $^{15}N$, for use in NMR spectroscopy.

Chemical shift dispersion

The range or spread of chemical shifts in an NMR spectrum; folded proteins show high dispersion, while unfolded proteins show lower dispersion.

Binding isotherm

A plot of chemical shift changes against ligand concentration used to calculate the binding affinity of a biomolecular interaction.

Coupled folding and binding

A binding model where an inherently unfolded protein, like pKID, folds into a specific structure (e.g., a helix) simultaneously as it interacts with its partner.

Conformational selection

A binding mechanism where a protein molecule samples different shapes and folds into its binding-competent form before it actually interacts with its partner.

J-couplings

Through-bond interactions used in NMR to transfer magnetisation between nuclei that are covalently bonded to one another.

2D ($^{1}H$, $^{15}N$) HSQC

A heteronuclear NMR experiment that produces a spectral 'fingerprint' with one crosspeak per NH group, representing individual residues in the protein.

Backbone resonance assignment

The process of identifying which specific residue in a polypeptide chain corresponds to each crosspeak in a 2D NMR spectrum.

HNCO experiment

A 3D NMR experiment that correlates the NH nuclei of residue $i$ with the carbonyl carbon (CO) nucleus of the preceding residue $i-1$.

Nuclear Overhauser effect (NOE)

A phenomenon that transfers magnetisation through space between nuclei separated by a distance of less than $6.0\,\text{\AA}$.

Root-mean-square deviation (RMSD)

A statistical value calculated for NMR structure ensembles where lower values indicate more precise or less flexible regions of the protein structure.

Spin-spin relaxation ($R_2$ or $T_2$)

An NMR parameter used to measure the rate ($R_2$) or time constant ($T_2$) of relaxation decay to monitor molecular dynamics on various time scales.