Robert W. Woody

[4] Circular dichroism

Publisher Summary Circular dichroism (CD) is a spectroscopic method which depends on the fact that certain molecules interact differently with right and left circularly polarized light. Circularly polarized light is chiral—that is, it occurs in two nonsuperimposable forms that are mirror images of one another. To discriminate between the two chiral forms of light, a molecule must be chiral, including the vast majority of biological molecules. A method that can discern the subtle differences between non superimposable mirror image molecules (enantiomers) must be highly sensitive to the three-dimensional features of molecules—that is, to conformation. Binding of ligands or protein-protein and protein–DNA interactions can also alter the circular dichroism spectrum of the protein and/or nucleic acid. These changes in CD can be used to determine equilibrium constants, and they can also provide evidence for conformational changes. Thus, CD can provide information about the secondary structure of proteins and nucleic acids and about the binding of ligands to these types of macromolecule.

Computation and analysis of protein circular dichroism spectra.

Publisher Summary This chapter presents computation and analysis of protein circular dichroism (CD) spectra. The origins of electronic CD in proteins, theoretical methods for computing protein CD, and empirical analysis of CD for estimating structural composition of proteins are described. The phenomenon of CD involves the absorption of light and it can be considered as a special type of absorption spectroscopy. The CD spectra of proteins are generally divided into three wavelength ranges, based on the energy of the electronic transitions that dominate in the given range. The basic approach used to compute the CD of complex systems, such as proteins and nucleic acids, is the divide and conquer approach. In addition to the isotropic atomic polarizabilities, the anisotropic polarizability of the first electrically allowed peptide transition was included in the dipole interaction model for protein CD calculations. Polarizability anisotropy data for simple amides were used for obtaining polarizability parameters. It is found that the CD spectrum of the typical β sheet has a negative band near 215-nm and a positive band near 198 nm.

Is polyproline II a major backbone conformation in unfolded proteins

Publisher Summary Protein folding is a process by which a polypeptide chain acquires its native structure from an unfolded state through a transition state. Recent studies of the unfolded states of proteins are based on a modification of the random coil model, recognizing that in many cases some residual native or non-native structure persists.. Combined evidence from the theoretical study of a blocked alanine peptide in aqueous solution and a variety of spectroscopic studies, including ultraviolet circular dichroism (CD), nuclear magnetic resonance (NMR), two-dimensional vibrational spectroscopy, vibrational circular dichroism (VCD), and vibrational Raman optical activity (VROA) reveal that the polyproline II (P II ) conformation is the dominant conformation in a variety of short model peptides. This chapter discusses the evidence from short peptides. It reviews the circular dichroism of unfolded proteins and addresses the role of P II in unfolded proteins.

Intrinsic protein disorder, amino acid composition, and histone terminal domains

Core and linker histones are the most abundant protein components of chromatin. Even though they lack intrinsic structure, the N-terminal “tail” domains (NTDs) of the core histones and the C-terminal tail domain (CTD) of linker histones bind to many different macromolecular partners while functioning in chromatin. Here we discuss the underlying physicochemical basis for how the histone terminal domains can be disordered and yet specifically recognize and interact with different macromolecules. The relationship between intrinsic disorder and amino acid composition is emphasized. We also discuss the potential structural consequences of acetylation and methylation of lysine residues embedded in intrinsically disordered histone tail domains.

Contributions of tryptophan side chains to the far-ultraviolet circular dichroism of proteins

It has often been assumed that the role of aromatic side chains in the far-ultraviolet region of protein circular dichroism (CD) is negligible. However, some proteins have positive CD bands in the 220–230 nm region which are almost certainly due to aromatic side chains. The contributions to the CD of interactions between tryptophan side chains and the nearest neighbor peptide groups have been studied, focusing on the indole Bb transition which occurs near 220 nm. Calculations on idealized peptide conformations show that the CD depends strongly on both backbone and side-chain conformation. Because of the low symmetry of indole, rotation about the CβCγ bond (dihedral angle χ2) by 180° generally leads to large changes in the CD, often causing the Bb band to reverse sign. When side-chain conformational preferences are taken into account, there is no strong bias for either positive or negative Bb rotational strengths. The observation that simple tryptophan derivatives such as N-acetyl-L-tryptophan methylamide have positive CD near 220 nm implies either that these derivatives prefer the αR region over the β region, or that there is little preference for χ2 < 180° over χ2 > 180°. Nearest-neighbor-only calculations on individual tryptophans in 15 globular proteins also reveal a small bias toward positive Bb bands. Rotational strengths of the Bb transition for some conformations can be as large as ∼ 1.0 Debye-Bohr magnetons in magnitude, corresponding to maximum molar ellipticities greater than 105 degcm2/dmol. Although a substantial amount of cancellation occurs in most of the examples considered here, such CD contributions could be significant, especially in proteins of low helix content.

On the analysis of membrane protein circular dichroism spectra

Analysis of circular dichroism spectra of proteins provides information about protein secondary structure. Analytical methods developed for such an analysis use structures and spectra of a set of reference proteins. The reference protein sets currently in use include soluble proteins with a wide range of secondary structures, and perform quite well in analyzing CD spectra of soluble proteins. The utility of soluble protein reference sets in analyzing membrane protein CD spectra, however, has been questioned in a recent study that found current reference protein sets to be inadequate for analyzing membrane proteins. We have examined the performance of reference protein sets available in the CDPro software package for analyzing CD spectra of 13 membrane proteins with available crystal structures. Our results indicate that the reference protein sets currently available for CD analysis perform reasonably well in analyzing membrane protein CD spectra, with performance indices comparable to those for soluble proteins. Soluble + membrane protein reference sets, which were constructed by combining membrane proteins with soluble protein reference sets, gave improved performance in both soluble and membrane protein CD analysis.

Comprehensive Chiroptical Spectroscopy: Applications in Stereochemical Analysis of Synthetic Compounds, Natural Products, and Biomolecules

This book provides an introduction to the important methods of chiroptical spectroscopy in general, and circular dichroism (CD) in particular, which are increasingly important in all areas of chemistry, biochemistry, and structural biology. The book can be used as a text for undergraduate and graduate students and as a reference for researchers in academia and industry. Experimental methods and instrumentation are described with topics ranging from the most widely used methods (electronic and vibrational CD) to frontier areas such as nonlinear spectroscopy and photoelectron CD, as well as the theory of chiroptical methods and techniques for simulating chiroptical properties. Applications of chiroptical spectroscopy to problems in organic stereochemistry, inorganic stereochemistry, and biochemistry and structural biology are also discussed, and each chapter is written by one or more leading authorities with extensive experience in the field.

Theory of Circular Dichroism of Proteins

In this chapter, the basic phenomenon of circular dichroism (CD) will be described. The central theoretical parameter of rotational strength will then be defined. The mechanisms by which electronic transitions contribute to CD, i.e., acquire rotational strength, will then be discussed qualitatively, after which the methods by which CD is calculated will be described. The most important group in the electronic spectroscopy of proteins, the peptide group, will then be discussed. Finally, theoretical studies of the principal types of peptide secondary structure will be surveyed. The reader should note that aromatic and disulfide groups are not discussed in this chapter, but are covered in a separate chapter (Woody and Dunker, Chapter 4), along with experimental studies of these important protein chromophores.

Aromatic and Cystine Side-Chain Circular Dichroism in Proteins

The contributions of aromatic side chains to the near-UV CD spectra of proteins are widely recognized and utilized as sensitive probes of protein conformation and ligand binding. The analysis of the near-UV CD spectra of proteins has been reviewed by Strickland (1974), Kahn (1979), and Drake (1993). Applications to studies of ligand binding were reviewed by Greenfield (1975). Our own computer-aided literature searches indicate that well over 600 papers have reported usage of near-UV CD spectra in recent years. Space and time limitations preclude a comprehensive review of these studies. In the limited review presented here, we have attempted to provide a sense of the range of the applications, with no claim that the “best” or “most useful” papers have been selected.

Structural composition of βI‐ and βII‐proteins

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of α‐rich proteins are similar to those of model α‐helices, but β‐rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model β‐sheets or unordered polypeptides. The existence of these two types of CD spectra for β‐rich proteins form the basis for their classification as βI‐ and βII‐proteins. Although the conformation of β‐sheets is largely responsible for the CD spectra of βI‐proteins, the source of βII‐protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II‐type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the β‐sheet and P2 structure contents in β‐rich proteins to understand the origin of βII‐protein CD. We find that βII‐proteins have a ratio of P2 to β‐sheet content greater than 0.4, whereas for βI‐proteins this ratio is less than 0.4. The β‐sheet content in βI‐proteins is generally higher than that in βII‐proteins. The origin of two classes of CD spectra for β‐rich proteins appears to lie in their relative β‐sheet and P2 structure contents.