Norma J. Greenfield

Using circular dichroism spectra to estimate protein secondary structure

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 ≤20 μg 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.

[27] Circular dichroism and optical rotatory dispersion of proteins and polypeptides

Publisher Summary This chapter discusses the methodology of circular dichroism (CD) and optical rotatory dispersion (ORD) data gathering and analysis in the rapidly changing field of protein structure, making use of synthetic polypeptide studies when necessary. ORD is the measurement, as a function of wavelength, of a molecules ability to rotate the plane of linearly polarized light. CD is similar data evaluating the molecules unequal absorption of right- and left-handed circularly polarized light. CD and ORD can yield useful estimates of protein secondary structure. Although all the amino acids except glycine contain at least one asymmetric carbon atom, most amino acids display only small ORD and CD bands. It is the conformation of the protein—that is, the asymmetric and periodic arrangement of peptide units in space, which gives rise to their characteristic ORD and CD spectra. In recent years, X-ray diffraction analysis has lead to the complete mapping of the peptide backbone and side-chain positions of lysozyme, several other enzymes, and quite a few other proteins in the solid state.

Deciphering the design of the tropomyosin molecule

The crystal structure at 2.0-Å resolution of an 81-residue N-terminal fragment of muscle α-tropomyosin reveals a parallel two-stranded α-helical coiled-coil structure with a remarkable core. The high alanine content of the molecule is clustered into short regions where the local 2-fold symmetry is broken by a small (≈1.2-Å) axial staggering of the helices. The joining of these regions with neighboring segments, where the helices are in axial register, gives rise to specific bends in the molecular axis. We observe such bends to be widely distributed in two-stranded α-helical coiled-coil proteins. This asymmetric design in a dimer of identical (or highly similar) sequences allows the tropomyosin molecule to adopt multiple bent conformations. The seven alanine clusters in the core of the complete molecule (which spans seven monomers of the actin helix) promote the semiflexible winding of the tropomyosin filament necessary for its regulatory role in muscle contraction.

Applications of circular dichroism in protein and peptide analysis

Abstract This review discusses several useful applications of circular dichroism as a tool for analyzing properties of proteins. The following topics are discussed: (1) protein–ligand interactions; (2) thermodynamics of protein folding; (3) conformational transitions and protein aggregation; (4) folding intermediates; (5) kinetics of protein folding. Specific examples are given to illustrate each application.

Disease-causing mutations in cardiac troponin T: identification of a critical tropomyosin-binding region.

Fifteen percent of the mutations causing familial hypertrophic cardiomyopathy are in the troponin T gene. Most mutations are clustered between residues 79 and 179, a region known to bind to tropomyosin at the C-terminus near the complex between the N- and C-termini. Nine mutations were introduced into a troponin T fragment, Gly-hcTnT(70-170), that is soluble, alpha-helical, binds to tropomyosin, promotes the binding of tropomyosin to actin, and stabilizes an overlap complex of N-terminal and C-terminal tropomyosin peptides. Mutations between residues 92 and 110 (Arg92Leu, Arg92Gln, Arg92Trp, Arg94Leu, Ala104Val, and Phe110Ile) impair tropomyosin-dependent functions of troponin T. Except for Ala104Val, these mutants bound less strongly to a tropomyosin affinity column and were less able to stabilize the TM overlap complex, effects that were correlated with increased stability of the troponin T, measured using circular dichroism. All were less effective in promoting the binding of tropomyosin to actin. Mutations within residues 92-110 may cause disease because of altered interaction with tropomyosin at the overlap region, critical for cooperative actin binding and regulatory function. A model for a five-chained coiled-coil for troponin T in the tropomyosin overlap complex is presented. Mutations outside the region (Ile79Asn, Delta 160Glu, and Glu163Lys) functioned normally and must cause disease by another mechanism.

Analysis of circular dichroism data

Publisher Summary This chapter focuses on the analysis of circular dichroism (CD) data to determine thermodynamic parameters of folding, binding constants, and estimates of secondary structure. Proteins and polypeptides have CD bands in the far ultraviolet region that arise mainly from the amides of the protein backbone and are sensitive to their conformations. Proteins have CD bands in the near ultraviolet and visible regions, which arise from aromatic amino acids and prosthetic groups. CD can be used to determine the enthalpy, entropy and midpoints, and values of unfolding/refolding transitions of a protein if they are reversible as a function of temperature or denaturant. The change in CD as a function of ligand concentration has been used to study numerous systems. It is found that if two proteins bind to each other only when they are folded and the protein complex unfolds cooperatively and reversibly to give two unfolded monomers, it is easy to determine the binding constant by determining the thermodynamics of folding of the complex compared with the thermodynamics of folding of the monomers.

Haploinsufficiency of MSX1: a Mechanism for Selective Tooth Agenesis

ABSTRACT Previously, we found that the cause of autosomal dominant selective tooth agenesis in one family is a missense mutation resulting in an arginine-to-proline substitution in the homeodomain of MSX1. To determine whether the tooth agenesis phenotype may result from haploinsufficiency or a dominant-negative mechanism, we have performed biochemical and functional analyses of the mutant protein Msx1(R31P). We show that Msx1(R31P) has perturbed structure and reduced thermostability compared with wild-type Msx1. As a consequence, the biochemical activities of Msx1(R31P) are severely impaired, since it exhibits little or no ability to interact with DNA or other protein factors or to function in transcriptional repression. We also show that Msx1(R31P) is inactive in vivo, since it does not display the activities of wild-type Msx1 in assays of ectopic expression in the limb. Furthermore, Msx1(R31P) does not antagonize the activity of wild-type Msx1 in any of these assays. Because Msx1(R31P) appears to be inactive and does not affect the action of wild-type Msx1, we propose that the phenotype of affected individuals with selective tooth agenesis is due to haploinsufficiency.

Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism

Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments require 2–5 h.

Tropomodulin Contains Two Actin Filament Pointed End-capping Domains

Tropomodulin 1 (Tmod1) is a ∼40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd ∼ 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd ∼ 0.1 μm). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160–359) contains the principal actin-capping activity (Kd ∼ 0.4 μm), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1–130) contains a second, weaker actin-capping activity (Kd ∼ 1.8 μm). Interestingly, 160–359 but not 1–130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.

Analysis of the kinetics of folding of proteins and peptides using circular dichroism.

Circular dichroism (CD) is a useful spectroscopic technique for studying the secondary structure, folding and binding properties of proteins. This protocol covers how to use the intrinsic circular dichroic properties of proteins to follow their folding and unfolding as a function of time. Included are methods of obtaining data and for analyzing the folding and unfolding data to determine the rate constants and the order of the folding and unfolding reactions. The protocol focuses on the use of CD to follow folding when it is relatively slow, on the order of minutes to days. The methods for analyzing the data, however, can also be applied to data collected with a CD machine equipped with stopped-flow accessories in the range of milliseconds to seconds and folding analyzed by other spectroscopic methods including changes in absorption or fluorescence spectra as a function of time.