Spin-Label Electron Paramagnetic Resonance Spectroscopy

Derek Marsh

• Publisher: CRC Press
• Year: 2019
• ISBN:
  9780429528675
• Edition: 1
• Topic: Medicina
• Subtopic: Bioquímica en medicina

Spin-label electron paramagnetic resonance (EPR) spectroscopy is a versatile molecular probe method that finds wide application in molecular biophysics and structural biology. This book provides the first comprehensive summary of basic principles, spectroscopic properties, and use for studying biological membranes, protein folding, supramolecular structure, lipid-protein interactions, and dynamics. The contents begin with discussion of fundamental theory and practice, including static spectral parameters and conventional continuous-wave (CW) spectroscopy. The development then progresses, via nonlinear CW-EPR for slower motions, to the more demanding time-resolved pulse EPR, and includes an in-depth treatment of spin relaxation and spectral line shapes. Once the spectroscopic fundamentals are established, the final chapters acquire a more applied character. Extensive appendices at the end of the book provide detailed summaries of key concepts in magnetic resonance and chemical physics for the student reader and experienced practitioner alike.

Key Features:

Indispensable reference source for the understanding and interpretation of spin-label spectroscopic data in its different aspects. Tables of fundamental spectral parameters are included throughout.

• Forms the basis for an EPR graduate course, extending up to a thorough coverage of advanced topics in Specialist Appendices. Includes all necessary theoretical background.

The primary audience is research workers in the fields of molecular biophysics, structural biology, biophysical chemistry, physical biochemistry and molecular biomedicine. Also, physical chemists, polymer physicists, and liquid-crystal researchers will benefit from this book, although illustrative examples used are often taken from the biomolecular field. Readers will be postgraduate researchers and above, but include those from other disciplines who seek to understand the primary spin-label EPR literature.

1

Introduction

1.1 Introduction

Electron paramagnetic resonance (EPR) – otherwise called electron spin resonance (ESR) – is a highly selective spectroscopy, because it requires an unpaired electron spin. In molecular systems, this restricts us to free radicals, transition metal compounds and the few paramagnetic molecules such as oxygen that have triplet ground states. Many free radicals are short-lived, and others are highly delocalized. Nitroxides, on the other hand, are stable free radicals in which the unpaired electron spin is localized to the N–O bond. Nearly all spin labels are nitroxides: N-oxyl derivatives of secondary amines, >N–O, in various nitrogen-containing heterocyclic rings. The nitroxide is protected against disproportionation reactions by immediately flanking methyl groups. This gives a simple EPR spectrum, where the hyperfine structure is dominated by the nitrogen nuclear spin.

Localization of the unpaired electron results in an angularly anisotropic spectrum that is an excellent reporter of molecular orientation and rotational dynamics. The full range of EPR properties that make nitroxides useful as spin labels is listed in Table 1.1. Linewidths (or T₂-relaxation) are determined by rapid rotational motions, in the nanosecond regime or faster. Power-saturation properties (or T₁-relaxation) are sensitive to slow rotation, with correlation times in the microsecond range. Absolute values of hyperfine couplings and g-values are sensitive to polarity of the immediate environment. The exchange interaction between spin labels can be used to measure collision rates and rates of translational diffusion. The magnetic dipole interaction between spin labels, or between spin labels and transition metal ions, can be used to measure distances in the range up to 8 nm, or possibly further.

Nitroxide free radicals are not only inherently stable but also can participate in a versatile chemistry. This contributes greatly to their value as spin labels. Figure 1.1 shows some typical spin labels: these divide themselves into small molecules, which are used as dilute probes, and nitroxide-bearing reagents that can be used for labelling macromolecules. The probes are either simple soluble nitroxides or small molecules such as lipids, ligands and enzyme substrates that are spin-labelled. Prominent among the spin-label reagents are those for covalently modifying protein side chains. These are analogues of the classical reagents of protein chemistry. Although many applications of spin labelling are in the fields of molecular biophysics and structural biology, these EPR techniques are equally applicable to other areas of physical and macromolecular chemistry, soft-matter physics and materials science.

TABLE 1.1 EPR properties of spin labels

FIGURE 1.1 Nitroxide spin labels. TEMPO derivatives substituted at 4-position of piperidinyl ring; PROXYL derivatives at 3-position of pyrrolidinyl ring; PY derivatives at 3-position of pyrrolinyl ring; DOXYL derivatives at 2-position of oxazolidinyl ring. See Table A1.1 for chemical names.

The nitroxide moieties in Figure 1.1 fall into a few major categories: the six-membered piperidine ring (TEMPO), the five-membered pyrrolidine (PROXYL) and pyrroline (PY) rings, and the five-membered oxazolidine ring (DOXYL). For spin labelling, the 6-membered rings normally are substituted at the 4-position, the five-membered pyrrolidine and pyrroline rings at the 3-position (where the nitroxyl is at the 1-position) and the oxazolidine ring at the 2-position (where the nitroxyl is at the 3-position). Therefore, we can usefully name spin-labelled compounds as: 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl (TEMPO), 1-oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl (PROXYL), 1-oxyl-2,2,5,5- tetramethylpyrrolin-3-yl (PY) and ...