Backbone assignment and secondary structure of the PsbQ protein from Photosystem II

Abstract

PsbQ is one of the extrinsic proteins situated on the lumenal surface of photosystem II (PSII) in the higher plants and green algae. Its three-dimensional structure was determined by X-ray crystallography with exception of the residues 14-33. To obtain further details about its structure and potentially its dynamics, we approached the problem by NMR. In this paper we report (1)H, (15)N, and (13)C NMR assignments for the PsbQ protein. The very challenging oligo-proline stretches could be assigned using (13)C-detected NMR experiments that enabled the assignments of twelve out of the thirteen proline residues of PsbQ. The identification of PsbQ secondary structure elements on the basis of our NMR data was accomplished with the programs TALOS+, web server CS23D and CS-Rosetta. To obtain additional secondary structure information, three-bond H(N)-H(α) J-coupling constants and deviation of experimental (13)C(α) and (13)C(β) chemical shifts from random coil values were determined. The resulting "consensus" secondary structure of PsbQ compares very well with the resolved regions of the published X-ray crystallographic structure and gives a first estimate of the structure of the "missing link" (i.e. residues 14-33), which will serve as the basis for the further investigation of the structure, dynamics and interactions.

MQD—Multiplex‐Quadrature Detection in Multi‐Dimensional NMR

“…means that this peak could not be assigned) 26b. The 3D spectra were obtained in the same amount of time by acquiring a different number of data points in the indirect dimensions (TD1=54 and TD2=96 using the standard sequence and TD1=TD2=96 using MQD,).…”

Solution NMR and molecular dynamics reveal a persistent alpha helix within the dynamic region of PsbQ from photosystem II of higher plants

“…Cloning, overexpression and purification of uniformly doubly labeled 15 N, 13 C PsbQ from Spinacia oleracea in E. coli were described previously . The samples for NMR spectroscopy contained 800 µM 15 N, 13 C PsbQ, 20 m M Na 2 HPO 4 , 50 µM NaN 3 , 1 m M EDTA (pH = 7.0) in H 2 O: 2 H 2 O 90:10.…”

“…Since this cross‐linked site of PsbQ is not visible in the electron density map, it was supposed that the N‐terminal stretch of free PsbQ is very flexible and probably extended in solution . Previous NMR studies of the secondary structure of PsbQ in solution indicated some residual order in the so far unresolved dynamic N‐terminal region which was however inconsistent with the short parallel β‐sheet (I5‐V7 and F38‐L40) reported in the crystallographic structure …”

Proline Fingerprint in Intrinsically Disordered Proteins

“…Carbon‐13 direct‐detection NMR has proven to be very valuable for studying IDPs/IDRs . In particular, the detection of carbonyl carbon atoms (C′) constitutes a particularly useful tool for detection of proline residues . In this category, the simplest yet most effective experiment is the 2D CON in which the correlations between backbone carbonyl carbon and nitrogen of neighboring residues are detected .…”