Proton magnetic resonance spectroscopic studies using paramagnetics of primary and tertiary structure of proteins

Abstract

On binding a paramagnetic broadening probe at the end of a random coil polypeptide chain, one obtains sequential broadening of the -CH resonances along the chain and, hence, can determine the sequence of the peptide. The method has been applied to tripeptides (0.25 mg of material being used), tetrapeptides and a hexapeptide under conditions such that gadolinium ions bind at the C-terminus or cupric ions at the N-terminus.Dimethylation of the lysine residues of lysozyrne and observation of the chemical shifts of the six dimethyl proton resonances as a function of pH allows the determination of their pK. From studies of selective broadening of the methyl resonances on addition of gadolinium ions and the known structure of the lysozyme-ion complex, it is possible to obtain the pK values of the lysine residues of lysozyme.Approaches to the determination of the structure in solution of proteins using paramagnetic broadening and shifting probes is discussed. Problems concerning the site of ion binding and the assignment of p.m.r. resonances arise when the crystal structure is unknown.The amount by which a proton magnetic resonance (p.m.r.) line is broadened by a paramagnetic probe with a long electronic relaxation time -e.g. gadolinium(III)-is dependent on the inverse sixth power of the distance between the nucleus giving rise to the resonance and the paramagnetic probe. Furthermore, if the paramagnetic probe is an ion which binds rapidly and reversibly at a specific site, then the width of the resonance line will progressively increase as the concentration of paramagnetic ion is increased until such time as the site is saturated. after which no further broadening will occur. On the binding of gadolinium ions at low concentration to lysozyme. there is preferential broadening of p.m.r. resonances that arise from protons which are closest to the binding 2 The binding site has been shown by x-ray studies3 to be located between glutamic acid 35 and aspartic acid 52. By the use of difference spectroscopy46, in this case the subtraction of a paramagnetically broadened spectrum from the spectrum of native lysozyme, it has been possible to observe the resonances from the side chains of valine 83

Introduction of a Strong Binding Site for Lanthanides at the N-Terminus of Peptides and Ribonuclease A

“…In a case such as that of lysozyme [12], where the structure and the binding site for lanthanides in the crystal is known, the problem is greatly simplified, since this information can be used for assignment of resonances [4,13]. In considering the problem of locating the position of a paramagnetic binding site in a protein of known sequence but of unknown three-dimensional structure, it seemed useful to develop chemical methods for the insertion of strong binding sites at specific locations in the molecule [2]. This would then solve the location problem, providing the introduced binding site was much stronger than any other in the molecule.…”

The lanthanide ions as structural probes in biological and model systems

“…Additional information is available on the following subjects: binding of Gd +a and Tb +3 to potentially bidentate monocarboxylates (226); structure and denticity of small peptides in solution (227) ; water-proton relaxation enhancement (228) and mapping studies of lysozyme (229); NMR investigation of yeast phosphoglycerate kinase (230) and its inhibition by lanthanide-ATP complexes (231) ; spin-label and lanthanide binding studies of glyceraldehyde-3-phosphate dehydrogenase (232); use of Pr +3 in the assignment of aromatic amino acid PMR resonances of horse ferricytochrome C (233); and primary sequencing of peptides (234,235).…”