Alice J. Adler

[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.

Conformational prediction and circular dichroism studies on the lac repressor

Abstract Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.

Complexes of deoxyribonucleic acid with lysine-rich (f1) histone phosphorylated at two separate sites: Circular dichroism studies

Abstract Samples of lysine-rich (f1) histone from calf thymus were enzymatically phosphorylated at each of two separate serine sites, one toward the N-terminus (ser-37), the other near the center of the molecule (ser-106). In addition, f1 phosphorylated simultaneously at both sites was prepared. Reconstituted complexes of calf thymus DNA with each of these phosphorylated histones were examined by means of circular dichroism. The conformational properties of these complexes were found to depend greatly upon the extent of f1 phosphorylation, but not significantly upon the position of phosphorylation. With each of the monophosphorylated histones, the magnitude of the circular dichroism changes of the DNA in f1-DNA complexes is diminished by approximately the same amount, as compared to nonphosphorylated f1-DNA. Furthermore, at any given f1: DNA ratio, complexes containing doubly phosphorylated f1 exhibit circular dichroic spectra more similar to that of free DNA than to the corresponding monophosphorylated complexes. Doubly phosphorylated f1 is particularly ineffective at inducing conformational distortion of DNA at high f1:DNA ratios. These observations cannot be attributed to reduced binding affinity to DNA for phosphorylated f1. The potential significance of multiple histone phosphorylation in modulating the function of f1 histone is discussed.

Rapid isolation of bovine interphotoreceptor retinol-binding protein

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent Mr is 250000 by gel-filtration chromatography, and 225000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent Mr is 140000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10(-6) M, as measured by fluorimetric titration.

Circular dichroism of valine and formylmethionine transfer RNA from Escherichia coli: Effect of aminoacylation

The ultraviolet circular dichroism spectra of two specific tRNAs, valine and formylmethionine-1, from Escherichia coli have been examined under various conditions. No significant changes were observed upon aminoacylation of either tRNA. Limits were placed upon possible differences in conformation (at most two base-pair hydrogen bonds and less than ten percent change in base stacking interactions) between the acylated and deacylated molecules. Differences are reported between valine and formylmethionine tRNAs. The circular dichroism of each tRNA species changes as a function of pH.

Purification and assay of interphotoreceptor retinoid-binding protein from the eye.

Publisher Summary Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular carrier of vitamin A and other lipids. This chapter discusses the purification procedures and assay of IRBP from the eye. The customary starting point for isolation of IRBP is a crude preparation of interphotoreceptor matrix (IPM). The IPM can be obtained by vigorous washing of the retina for maximum yield of IRBP; there is no need to avoid any tissue damage. Purification of IRBP to homogeneity consists of three chromatographic steps: Con A binding, anion exchange, and gel filtration. The purity of IRBP is assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with silver staining. The chapter presents purification of IRBP from six bovine eyes as an example. For small amounts of IRBP, especially in mixtures such as tissue samples, the only accurate methods are immunochemical.

Circular dichroism of Escherichia coli ribosomes: Effect of heating and metal ions

Abstract 1. Ribosomes and rRNA from a low-nuclease strain of Escherichia coli were examined by means of circular dichroism (CD) in 0.001 M Tris-acetate buffer (pH 7.2). 2. The ribosome CD spectrum was altered when metal ions were chelated with EDTA. The following changes occurred: disappearance of the small negative RNA band at 297 mμ, a red shift of the positive RNA band at 264 mμ and a slight decrease of the protein band at 222 mμ. Addition of Mg 2+ caused only partial reversal of these differences, and other metal ions including Ni 2+ had even smaller effects. 3. Similar irreversible CD changes were noted after heating and recooling the ribosomes. Hysteresis was evident in the heating and cooling curves. EDTA caused a lowering of the melting temperature. 4. The CD spectrum of isolated 30-S and 50-S ribosomes showed similar temperature effects. The CD spectra of the subunits differ in the protein region. 5. The CD spectrum of rRNA is dependent only upon metal ion concentration and not upon prior EDTA or heat treatment.