S Stob

PHOTO-CIDNP OF THE AMINO-ACIDS

Abstract

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.

AN EXCEPTION TO THE CIDNP SIGN RULES

Abstract It is demonstrated experimentally that the CIDNP sign rules can give erroneous results. Net effects observed in the 1 H NMR spectrum of 1-phenyl[2- 13 C]propan-2-one after photolysis are correctly predicted at 360 MHz but not at 100 MHz by the rules. The converse is true for the 1 H– 13 C multiplet effects. The radical pair theory can account for the CIDNP effects in all cases.

SURFACE ACCESSIBILITY OF AROMATIC RESIDUES IN HUMAN LYSOZYME USING PHOTOCHEMICALLY INDUCED DYNAMIC NUCLEAR-POLARIZATION NMR-SPECTROSCOPY

1H‐NMR CIDNP Lysozyme Surface accessibility

PHOTO-CIDNP IN GUANINE NUCLEIC-ACID DERIVATIVES - A MECHANISTIC STUDY

Abstract— A mechanistic study of the photo‐CIDNP effect of guanosine derivatives is presented. The pH dependence of the CIDNP effect of 5′‐GMP shows a sign reversal at pH 3.3. Using methylated guanosine derivatives evidence has been obtained that the protonation state of the N7 atom influences the radical pair generation. The mechanism involves a guanine radical dication (protonated radical cation) at low pH and a radical cation at high pH. The CIDNP behaviour of 5′‐GMP is compared with that of 5′‐AMP, that was previously studied.

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo‐CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His‐29 polarisations are weaker and broader in HP59 than in HP51 indicating that the C‐terminal octapeptide in HP59 adopts a conformation that allows an interaction with His‐29. The photo‐CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo‐excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo‐CIDNP effect of tyrosines 7 and 17 and abolishes the His‐29 polarisations. The results are compared with earlier photo‐CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece‐operator complex.

Magnetic field dependence of 1H CIDNP in the reaction of trimethyltin hydride with dibenzyl ketone

Abstract The magnetic field dependence of 1 H CIDNP in the photochemical reaction of trimethyltin hydride enriched by the stable isotope 117 Sn in the presence of dibenzyl ketone as initiator has been analysed. Comparing the experimental and simulated 1 H CIDNP spectra of benzyltrimethyltin shows that the radical pair mechanism can account for the observed CIDNP effects. The absence of a magnetic isotope effect in reactions of tin-containing compounds is discussed with reference to the obtained 1 H CIDNP data.

PHOTO-CIDNP STUDY OF ADENYLYL-CONTAINING DEOXY-DINUCLEOTIDES. PAIR SUBSTITUTION EFFECTS DUE TO INTRAMOLECULAR CATION RADICAL DEPROTONATION

Abstract— 360 MHz 1H photo‐CIDNP spectra of several adenylyl containing deoxy dinucleotides show that fast deprotonation at the 6‐amino group of the adenylyl radical cation can occur via two intramolecular routes. One is mediated by the 5′‐phosphomonoester group and only occurs for the pdA moiety at the 3′ end of the dinucleotide, e.g. in pdTpdA and pdApdA; molecular model building studies reveal that fluctuations away from the B‐DNA like conformation are involved. In the second route the deprotonation is catalyzed by N‐l located on an adjacent adenine ring, like in dApdA and pdApdA; it probably occurs for both adenylyl units in a stacked conformation. This fast deprotonation leads to the observation of negative polarization for the adenineH–8 as was observed in the case of adenosine 5‐monophosphate in the presence of added phosphate.