Brian D. Sykes

Fluorotyrosine alkaline phosphatase: internal mobility of individual tyrosines and the role of chemical shift anisotropy as a 19F nuclear spin relaxation mechanism in proteins.

19 F nuclear spin relaxation data for fluorotyrosine alkaline phosphatase at 94 and 235 MHz are presented. The factor of 2·5 in increased chemical shift separation expected at the higher field was almost exactly matched by an increase in 19 F linewidths due to the field-dependent relaxation mechanism of chemical shift anisotropy. Consequently, no significant improvement in resolution or signal/noise was gained at the higher field. At 235 MHz the chemical shift anisotropy mechanism produces about 50% of the observed linewidths but only 10 to 20% at 94 MHz. Using a density matrix formalism, complete expressions are derived for the relaxation of a nuclear spin with an asymmetric chemical shift tensor (three independent principal elements) undergoing axially symmetric (symmetric top) diffusion. With this formalism, the chemical shift anisotropy contributions to T 1 and T 2 for fluorine-labeled residues undergoing one link of internal motion may be calculated. Such a calculation requires detailed knowledge of both the magnitude and orientation within the molecular frame of the three principal elements of the traceless chemical shift tensor. Data from the literature on 19 F chemical shift tensors are collected and used in calculations of chemical shift anisotropy relaxation for m -fluorotyrosine, p -fluorophenylalanine, and trifluoromethyl residues incorporated in proteins. The relaxation is sensitive to internal motion about the C β −C γ bond in the aromatic residues and the C 3 axis of the CF 3 group. It is found that the contribution of chemical shift anisotropy to T 1 will be negligible except when dipolar relaxation is particularly inefficient and internal motion is near the Larmor frequence ω 0 . The contribution of chemical shift anisotropy to the linewidth increases with ω 0 2 and becomes significant for proteins with M r >20,000 and fields >50 kgauss. For fluorotyrosine alkaline phosphatase the calculated chemical shift anisotropy linewidths as a function of internal motion about C β −C γ span a range of values that encompasses the observed linewidths at 235 MHz for nine of the eleven fluorotyrosine residues. Two other fluorotyrosines exhibit linewidths with an even greater field dependence, suggesting the influence of chemical exchange ( via slow internal rotation) between different magnetic environments. Combining dipolar T 1 and linewidth data, chemical shift anisotropy linewidth data, and the nuclear Overhauser enhancement observed when irradiating protein protons, it is possible to derive the overall tumbling correlation time τ c for alkaline phosphatase and place limits on the internal mobility of the tyrosine residues. Thus, for overall tumbling we find that τ c =76·0±15 ns or D macro =2·2×10 6 s −1 . For rotation about C α −C β , D int ( α−β )≤10 6 s −1 for all tyrosines. Rotation about C β −C γ has D int ( β−γ )≤10 8 s −1 for all tyrosines, with nine tyrosines probably in the range 10 6 to 10 8 s −1 and two others in the range 10 2 to 10 5 s −1 . These results are compared with other literature data concerning protein mobility.

Dipolar nuclear spin relaxation of 19F in multispin systems. Application to 19F labeled proteins

A theoretical description of 19F–1H dipolar nuclear spin relaxation in multispin systems is presented which serves to explain the relaxation behavior of 19F in fluorine‐labeled proteins when the protons are not irradiated. The complete solution of the two‐spin 1H–19F spin–lattice relaxation is discussed for the various experimental pulse techniques commonly employed in Fourier transform NMR. Recovery curves following a single 90° pulse applied to 19F in spin systems containing several protons are simulated by using a digital computer to calculate the time development of the complete set of differential equations for z‐magnetizations. Steady‐state experiments such as progressive saturation were also examined. The simulated 19F relaxation curves for two‐spin and several‐spin systems (when the protons are not irradiated) are compared to the behavior expected if the relaxation followed an ’’ideal’’ single exponential with T1 given by the standard ’’unlike’’ spins formula. These calculations were performed for...

Design of stopped‐flow NMR rapid‐mixing cells

The design and construction of two rapid‐mixing apparatuses which can be accommodated within the probes of commercial NMR spectrometers are described. The principal features of the cells described are applicable to the construction of stopped‐flow cells for any NMR spectrometer.

THE DETECTION OF SUBSTRATE DISTORTION BY LYSOZYME: AN APPLICATION OF NMR TO THE STUDY OF ENZYME SUBSTRATE REACTIONS *

Fourier transform (FT) techniques have reduced the time required for the acquisition of an nmr spectrum to roughly the inverse of the resolution to be achieved in the final spectrum.l While this fact has focused attention on the possibility of using nmr to follow reaction kinetics, most applications of FT nmr have capitalized on this time saving to achieve a substantial improvement in sensitivity over continuous wave (CW) methods given the same amount of time. Nevertheless, the combination of the structural information available from nmr experiments with the time scale of rapid mixing experiments for the study of reacting systems has a great potential, especially in the observation of intermediates in chemical and biochemical reactions.? The great advantage of nmr over other detection methods (e.g., optical) commonly used in transient kinetic experiments is the type of detailed information available about each species whose appearance and/or disappearance is being followed during the reaction. The FT nmr techniques required for the study of reacting systems are described in this paper together with the application of these techniques to the study of the lysozyme-catalyzed cleavage of the tetrasaccharide (NAGNAM) ?

A nuclear magnetic resonance study of the binding of substrate analogues to a modified aspartate transcarbamylase.

from Micrococcus lysodeikticus.3* This reaction provides an example of a general class of reactions which can be represented by E + S % ES + E + P although the detailed mechanism in this example is more complex (see discussion below).5 Under conditions in which the exchange of the substrate between S and ES is rapid, so that the observed spectrum of the substrate is the weighted average of that for S and ES, and where the conversion of substrate to product is slow relative to the time required to acquire nmr spectra, the spectrum of the enzyme-bound substrate can be deduced without the ES complex having to be present at high enough concentration to be directly observable itself.6 In this manner the structure (in part) of the Iysozyme-bound tetrasaccharide has been obtained, and has thereby provided specific information relative to the structure of the intermediate in the lysozyme reaction mechanism. Lysozyme catalyzes the cleavage of cell wall oligosaccharides such as (NAG-NAM) Phillips and coworkers s-*? have proposed that the cleavage involves: ( 1 ) the binding of a hexasaccharide portion of the oligosaccharide

A nuclear magnetic resonance study of the binding of acetylsalicylic acid to human serum albumin.

Abstract Nuclear magnetic resonance methods have been used to determine the relationship between the modification of a specific residue in the active site of the enzyme aspartate transcarbamylase with a chromophoric mercurial and changes occurring in the binding of substrate analogs to the active site. The binding of succinate, an analog of the substrate L-aspartate, to the modified enzyme is completely blocked where as the binding of acetyl phosphate, an analog of the substrate carbamyl phosphate, is only slightly perturbed.

Kinetics of the interaction of methyl isonitrile with hemoglobin β chains: Measurement by nuclear magnetic resonance

Abstract The binding of acetylsalicylic acid to human serum albumin has been studied by nuclear magnetic resonance methods. The results indicate that acetylsalicylic acid exchanges rapidly with at least one site on human serum albumin, and that the nuclear spin relaxation of the acetyl protons of bound acetylsalicylic acid is characterized by a correlation time of the magnitude expected for the rotational correlation time of a human serum albumin molecule. The calculated rotational correlation time of the ASP·HSA complex is 4 × 10 −8 seconds. Limits are also set on the rate constants involved.

Water Eliminated Fourier Transform NMR Spectroscopy

Abstract The binding of methyl isonitrile (CH 3 Nandz.tbnd;C) to hemoglobin β chains has been studied by measuring the 1 H nuclear magnetic resonance transverse relaxation times for methyl isonitrile as a function of protein concentration, temperature and 14 N decoupling. Binding of methyl isonitrile both at the heme iron and at a non-specific site (or sites) has an effect upon the measured nuclear spin relaxation times. The results yield a value of 57 ± 12 seconds −1 (20 °C) for the “off” rate constant K −1 for specific binding and an Arrhenius activation energy for k −1 of 14 ± 3 kcal mol −1 .