BIOL5372M - Advanced Biomolecular Technologies - University of Leeds

Biological NMR data analysis

Task 1: 2D NMR

Problem 1:

The figures below show three amide HSQC spectra, each recorded on one of three protein samples:

(i) The 15N labelled human cellular retinoic acid binding protein 1 (CRABP1), a 14 kDa globular protein;;

(ii) The 2H,15N labelled nucleotide-­binding domain of the E. coli Hsp70 molecular chaperone (NBD), a 44 kDa globular protein

(iii) The 15N cytoplasmic domain of the T cell receptor zeta chain (ZETA), a 10 kDa intrinsically disordered protein

Compare the amide HSQC spectra and identify the protein for each protein sample. Briefly explain your choice.

Task 2: 3D NMR

Problem 2:

The following two-­dimensional spectra are slices (strips) from a HNCO experiment at the indicated 15N frequencies.
(i) Write chemical shifts (CSs) for individual 1HN, 15NH and carbonyl 13C atoms. For example:
(ii) Label peaks in the HSQC spectrum below (i.e. T64, E119, etc):
(iii) The HSQC and HNCO spectra have the same proton (1H) and nitrogen (15N) dimensions. Explain why some peaks that overlap in the HSQC spectrum occur as separate peaks in the HNCO spectrum.

Task 3: NMR peak assignments

Problem 3:

To assign protein B from task 1, the 3D CACBNH and CBCA(CO)NH were recorded. The analysis of the NMR spectra revealed several segments of linked residues;; including five sequentially connected residues, labelled as A-­E in the 2D HSQC spectrum below:

Using the 2D strips from CACBNH (labelled i) and CBCA(CO)NH (labelled ii) experiments at indicated nitrogen frequencies for five peaks from the 2D HSQC spectrum

-­ write chemical shifts (CSs) for individual 1HN, 15NH and 13C atoms for each residue;;
-­ if possible, identify the type of amino acid;;
-­ determine the order of these five strips;;
-­ assign them to the protein primary sequence:

MGHHHHHHHHHHSSGHIEGRHMPNFAGTWKMRSSENFDELLKALGVNAMLRKVAVAAASKPHV EIRQDGDQFYIKTSTTVRTTEINFKVGEGFEEETVDGRKCRSLPTWENENKIHCTQTLLEGDGPKT YWTRELANDELILTFGADDVVCTQIYVRE

Task 4: Ligand binding

Problem 4:

Hsp70 chaperones are key hubs in cellular proteostasis and protein quality control networks in the cytoplasm and organelles. They facilitate protein folding, disaggregation of misfolded proteins, protein degradation, protein refolding and assembly of protein complexes. Hsp70 functions and activity rely on ATP binding to the Hsp70 N-­terminal nucleotide-­binding domain (NBD) to allosterically alter substrate affinity to the C-­terminal substrate-­binding domain (SBD). Initial screening of the ER Hsp70 molecular chaperone BiP reveals that the compound A weakly interacts with the protein (Kd=100 μM). The analysis of NMR chemical shift perturbations revealed a set of residues with significant changes in 1H chemical shifts of BiP when 5 mM of the compound A was added to 100 μM of 2H,15N labelled BiP:

-­ Residues with large chemical shift changes (Δδ1HN ?> ?0.5 ?ppm) upon A binding to BiP: Gly36, Thr37, Thr38, Tyr39, Gly226, Gly227, Gly255, Lys296, Arg297, Ala298, Ser300, Gly363, Gly364,
-­ Residues with moderate chemical shift changes (Δδ1HN ?> ?0.05 ?ppm):
Ser40, Gly228, Ala229, Glu256, Glu293, Lys294, Ala295, Leu299, Ser301, Ser365, Arg367, Ile368, Gln373, Asp391.
-­ Residues with small significant chemical shift changes (Δδ1HN ?> ?0.02 ?ppm):
Asp34, Leu35, Cys41, Ser64, Lys96, Val172, Glu201, Asp224, Leu225, Phe230, Gly254, Phe258, Asp259, Val292, Gln302, Lys340, Vla362, Thr366, Gly392.

To design improved inhibitors of BiP a pharmaceutical company initiates a programme of structure-­ based drug design. Use the chemical shift perturbations from the table below to map the binding site for the compound A on the BiP structure (PDB ID: 5E84, chain A). Suggest a plausible molecular mechanism(s) of how compound A can interfere with BiP function. In order to answer this Problem you will need to examine the structure of ATP-­bound BiP (PDB ID: 5E84, chain A). This should be done using the Pymol structure visualisation software.

A set of compounds related to A were synthesised and investigated by NMR. The NMR data for three new compounds are presented below. Figure shows the dependence of NMR peak positions on the compound concentrations for a representative residue (Gly227)

Estimate (roughly, you don't need to do calculations) where the compounds B, C and D are more stronger or weaker binders? Explain your suggestions. Which is the ‘better drug'?

Attachment:- Biological NMR data analysis.rar