Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that measurements may be made on multiple samples containing < or =20 microg of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by x-ray crystallography or NMR.

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Using circular dichroism spectra to estimate protein secondary structure

https://www.cell.com/cell/pdf/S0092-8674(00)80205-6.pdf

Core structure of GP41 from the HIV envelope glycoprotein

A new method is developed in which a circular dichroism (CD) spectrum is analyzed directly as a linear combination of the CD spectra (from 190 to 240 nm) of 16 proteins whose secondary structures are known from X-ray crystallography. This avoids the dilemma encountered in previous methods of trying to define single reference CD spectra that were supposed to characterize such broad and variable classes as helix, beta sheet, beta turn, and "remainder". It also permits a more accurate and flexible analysis. The usual instability in using so many parameters is automatically controlled by a simple constrained statistical regularization procedure (similar to ridge regression). Sixteen tests were made by removing 1 spectrum at a time from the set of 16 and analyzing it in terms of the other 15. The product moment correlation coefficients between the computed fractions of helix, beta sheet, beta turn, and remainder and the fractions from the X-ray data were 0.96, 0.94, 0.31,, and 0.49, respectively. Thus, the helix and beta-sheet accuracy is very good. (The corresponding values calculated by a previous method with four reference spectra were 0.85, 0.25, --0.31, and 0.46.).

Estimation of globular protein secondary structure from circular dichroism.

Publisher Summary This chapter discusses some recent developments in the estimation of various conformations in a protein molecule from its circular dichroism (CD) spectrum in the ultraviolet region Optical rotatory dispersion (ORD) and CD are two chiroptical phenomena, which differentiate two enantiomers They are caused by different interactions of left- and right-circularly polarized light with chiral molecules Chirality is a geometric property of molecules; the corresponding substances are therefore optically active At present CD has replaced ORD for the conformational analysis of proteins because CD bands that are characteristic of various secondary structures can be directly observed The very simplicity of the CD method and the short time required for the measurements are extremely attractive It is often advantageous to compare the CD analysis with empirical predictions of secondary structure of proteins from sequences, if such are available All current methods for CD analysis are developed to determine not only the helix and β-form but also the β-turn and unordered forms

Calculation of protein conformation from circular dichroism.

Gene-encoded antimicrobial peptides are an important component of host defense in animals ranging from insects to mammals. They do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. They are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. A particularly abundant and widespread class of antimicrobial peptides are those with amphipathic, alpha-helical domains. Due to their relatively small size and synthetic accessibility, these peptides have been extensively studied and have generated a substantial amount of structure-activity relationship (SAR) data. In this review, alpha-helical antimicrobial peptides are considered from the point of view of six interrelated structural and physicochemical parameters that modulate their activity and specificity: sequence, size, structuring, charge, amphipathicity, and hydrophobicity. It begins by providing an overview of how these vary in peptides from different natural sources. It then analyzes how they relate to the currently accepted model for the mode of action of alpha-helical peptides, and discusses what the numerous SAR studies that have been carried out on these compounds and their analogues can tell us. A comparative analysis of the many alpha-helical, antimicrobial peptide sequences that are now available then provides further information on how these parameters are distributed and interrelated. Finally, the systematic variation of parameters in short model peptides is used to throw light on their role in antimicrobial potency and specificity. The review concludes with some considerations on the potentials and limitations for the development of alpha-helical, antimicrobial peptides as antiinfective agents.

Kue Hung Chau

Papers

Determination of the helix and beta form of proteins in aqueous solution by circular dichroism.