Klaas Dijkstra

Sequential assignment of imino- and amino-proton resonances in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy. Application to a lac operator fragment

A sequential procedure, based on 2D NOE spectroscopy, is described for identifying the resonances of water-exchangeable imino protons in NMR spectra of oligonucleotides. The adenine H2 and amino protons are also part of the cross-relaxation network of the imino protons and their resonance frequencies are easily found in the 2D NOE spectra. The NOE contacts between the amino and H5 protons of cytidines form an independent check for the correctness of the assignments, since these CHS protons are also part of the cross-relaxation network formed by the nonexchangeable protons, for which another sequential-assignment procedure exists (R. M. Scheek et al., J. Am. Chem. Soc.14, 2914 (1983)). The method was applied to a 14 bp lac operator fragment. The assignments of the resonances of the water-exchangeable imino and amino protons in this DNA fragment are required for studying its complex with the lac repressor headpiece.

The structure in solution of the b domain of protein disulfide isomerase

Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b′ and a′. Both a and a′ domains contain an active site with the sequence motif -Cys-Gly-His-Cys- involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol., 7, 239–245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.

The NMR determination of the IIAmtl binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system

The region of the surface of the histidine‐containing protein (HPr) which interacts with the A domain of the mannitol‐specific Enzyme II (IIAmtl) has been mapped by titrating the A‐domain into a solution of 15N‐labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty‐five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of IIAmtl while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of IIAmtl resulted in chemical shift changes in a sub‐set of the above residues; these were located more in the vicinity of the active site phospho‐histidine. Phosphorylation of the HPr/IIAmtl complex resulted in a HSQC spectrum which was indistinguishable from the P‐HPr spectrum in the absence of IIAmtl indicating that, as expected, the complex P‐HPr/P‐IIAmtl does not exist even at the high concentrations necessary for NMR.

Role of aromatic amino acids in carbohydrate binding of plant lectins: Laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins

Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair‐generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light‐absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP‐reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein‐carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N‐acetylglucosamine‐binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR‐ or X‐ray crystallographic data permit a decision of the validity of the CIDNP method‐derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein‐carbohydrate interactions in solution, complementing methods such as X‐ray crystallography, high‐resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling. Proteins 28:268–284, 1997

FLASH-PHOTOLYSIS NMR - CIDNP TIME-DEPENDENCE IN CYCLIC PHOTOCHEMICAL-REACTIONS

Abstract A microsecond NMR flash photolysis technique has been used to study the tune dependence of CIDNP spectra observed during the cyclic photochemical reaction of N-acetyltryptophan with 3-N-carboxy methyllumiflavin. The rapid fall in the flavin enhancements is attributed to cancellation of escape- and recombination-type polarizations. This is caused by the dispropornonation of flavin radicals.

Graphical interpretation of Boolean operators for protein NMR assignments

We have developed a graphics based algorithm for semi-automated protein NMR assignments. Using the basic sequential triple resonance assignment strategy, the method is inspired by the Boolean operators as it applies “AND”-, “OR”- and “NOT”-like operations on planes pulled out of the classical three-dimensional spectra to obtain its functionality. The method’s strength lies in the continuous graphical presentation of the spectra, allowing both a semi-automatic peaklist construction and sequential assignment. We demonstrate here its general use for the case of a folded protein with a well-dispersed spectrum, but equally for a natively unfolded protein where spectral resolution is minimal.

1H-NMR and photochemically-induced dynamic nuclear polarization studies on bovine pancreatic phospholipase A2

Proton-NMR resonances of trytophan 3 and tyrosine 69 in bovine pancreatic phospholipase A2, its pro-enzyme and in Ala1-transaminated protein were assigned using photochemically-induced dynamic nuclear polarization (photo-CIDNP) as such or in combination with spin-echo measurements. In addition assignments were made by suppression of cross-relaxation effects using short (0.1 s) high-power laser pulses.

Order-parameter tensor description of HPr in a medium of oriented bicelles

Residual dipolar couplings between 15N and 1H nuclear spins in HPr were used to determine the proteins orientation in a medium of bicelles, oriented by a magnetic field. In the case of wild-type HPr the proteins non-spherical shape can explain its orientation in this medium. In the case of the F48W mutant it was found that at least one other mechanism contributes to the observed orientation of the protein, to a degree that depends on the concentration of phosphate ions in the medium. We propose that the F48W mutant has a weak affinity towards the bicelle-surfaces that decreases with increasing phosphate concentrations. We used an order-parameter description to analyse this situation and to determine the axis of main order and the sign of the order parameter pertaining to this additional orientation mechanism.

Applications of two-dimensional 1H nuclear magnetic resonance methods in photochemically induced dynamic nuclear polarisation spectroscopy

Photochemically induced dynamic nuclear polarisation (photo-CIDNP) has been widely used to identify surface residues in proteins and to assign resonances in their 1H n.m.r. spectra by the spectral simplification that is obtained. Welcome as this simplification may be in many cases, it also presents a limitation to the technique. We have increased the information content of photo-CIDNP spectra using pulse sequences analogous to INEPT (CIDNPT and refocused CIDNPT). Furthermore, we present combinations of photo-CIDNP with two-dimensional (2D)J-correlated spectroscopy (CIDNP-COSY) and with 2D nuclear Overhauser effect spectroscopy (CIDNP-NOESY). In these experiments we insert a saturation pulse sequence and a short laser-irradiation period at the beginning of the preparation period to generate CIDNP, leaving the remainder of the pulse scheme essentially unaltered.The CIDNPT technique has been applied to N-acetyl tyrosine and to N-acetyl tryptophan. The CIDNP-COSY spectrum of the lac-repressor headpiece is presented. The CIDNP-NOESY spectrum of hen egg-white lysozyme is compared with 1 D cross-polarisation spectra of the same protein.

Solution NMR Study of the Monomeric Form of p13 suc1 Protein Sheds Light on the Hinge Region Determining the Affinity for a Phosphorylated Substrate

Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal β-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13 suc1 CKS protein fromSchizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the β-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13 suc1 and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal β-sheet involved in domain swapping and protein interactions.