W. Curtis Johnson

Analyzing protein circular dichroism spectra for accurate secondary structures

We have developed an algorithm to analyze the circular dichroism of proteins for secondary structure. Its hallmark is tremendous flexibility in creating the basis set, and it also combines the ideas of many previous workers. We also present a new basis set containing the CD spectra of 22 proteins with secondary structures from high quality X‐ray diffraction data. High flexibility is obtained by doing the analysis with a variable selection basis set of only eight proteins. Many variable selection basis sets fail to give a good analysis, but good analyses can be selected without any a priori knowledge by using the following criteria: (1) the sum of secondary structures should be close to 1.0, (2) no fraction of secondary structure should be less than –0.03, (3) the reconstructed CD spectrum should fit the original CD spectrum with only a small error, and (4) the fraction of α‐helix should be similar to that obtained using all the proteins in the basis set. This algorithm gives a root mean square error for the predicted secondary structure for the proteins in the basis set of 3.3% for α‐helix, 2.6% for 310‐helix, 4.2% for β‐strand, 4.2% for β‐turn, 2.7% for poly(L‐proline) II type 31‐helix, and 5.1% for other structures when compared with the X‐ray structure. Proteins 1999;35:307–312.

Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication

Inverse circular dichroism (CD) spectra are presented for each of the five major secondary structures of proteins: alpha-helix, antiparallel and parallel beta-sheet, beta-turn, and other (random) structures. The fraction of the each secondary structure in a protein is predicted by forming the dot product of the corresponding inverse CD spectrum, expressed as a vector, with the CD spectrum of the protein digitized in the same way. We show how this method is based on the construction of the generalized inverse from the singular value decomposition of a set of CD spectra corresponding to proteins whose secondary structures are known from X-ray crystallography. These inverse spectra compute secondary structure directly from protein CD spectra without resorting to least-squares fitting and standard matrix inversion techniques. In addition, spectra corresponding to the individual secondary structures, analogous to the CD spectra of synthetic polypeptides, are generated from the five most significant CD eigenvectors.

A circular dichroism spectrometer for the vacuum ultraviolet.

We have developed a circular dichroism spectrometer which will make measurements in the ultraviolet to 1350 A. It is built primarily from commercially available components and is particularly simple in design. The instrument is linear and has a noise level over most of its range of less than 1×10−5 OD units for a 16 A spectral slitwidth and 10 sec time constant. The absorption and circular dichroism spectra of (+) 3‐methylcyclopentanone to 1350 A are presented.

Extending CD spectra of proteins to 168 nm improves the analysis for secondary structures.

The CD spectra for 10 proteins with known secondary structure have been extended from 178 to 168 nm. Combined with the data for 6 other proteins investigated previously, this produces a basis set of 16 proteins, which can be used to analyze CD spectra for secondary structure. Extending the spectra adds another CD band to the data and increases the information content from the equivalent of five to six. Analyzing the CD for each of the 16 proteins in the basis set with the 15 other proteins shows a modest improvement in the prediction of secondary structure with the extended CD spectra.

Experimental errors and their effect on analyzing circular dichroism spectra of proteins.

Abstract Seven types of error that may interfere with the analysis of protein circular dichroism (CD) spectra for secondary structure are examined. Three of these errors are operational encompassing wavelength synchronization, and proper choice of spectral bandwidth and scan speed. Three are experimental involving intensity adjustments and two sources of baseline shift. The skew baseline shift is analogous to error in CD intensity at short wavelengths due to high sample absorption and low source intensity. The final source of error deals with constrained analyses. We have investigated these types of error to determine how they may be affecting our analysis of protein CD spectra and the role they may play in causing our analyses to fail for some proteins. We find that small errors in the baseline (which are independent of the protein spectrum) will rationalize our poor analyses. Spectroscopists must adopt new standards of precision if sophisticated analyses are to succeed.

Assigning secondary structure from protein coordinate data

We have developed a program to convert the three dimensional coordinates describing protein structure in the Brookhaven Data Bank into an assignment of secondary structure. The program assigns secondary structure in the same way a person assigns structure visually. It uses two angles and three distances to assign α‐helix, 310‐helix, β‐strand, hydrogen‐bonded β‐turn, non‐hydrogen‐bonded β‐turn, and poly (L‐proline) II type 31‐helix. The program is concerned with amide‐amide interactions and should be particularly useful to spectroscopists. Proteins 1999;35:313–320.

Conformation of DNA in ethylene glycol

Abstract Detailed circular dichroism studies lead us to propose that the low temperature form of DNA in ethylene glycol is the C type conformation. We confirm that DNA in high salt is intermediate between B and C form and suggest that the high temperature form of DNA in ethylene glycol is a “random coil”. Speculation on C form DNA as an important in vivo conformation is given in the conclusion. The DNA-ethylene glycol system offers a convenient way to study this structure.

Determination of the Conformation of Nucleic Acids by Electronic CD

The natural and synthetic nucleic acids are polymers of nucleotides that in turn are made up of an aromatic base, a sugar, and a phosphate group. The bases are the chromophores that absorb ultraviolet light to undergo electronic transitions, which begin at 300 nm and continue into the vacuum UV region. In the case of DNA these bases are adenine (A), guanine (G), cytosine (C), and thymine (T); in the case of RNA the bases are A, G, C, and uracil (U), which is closely related to T both structurally and chromophorically. The structure of these five bases is given in Fig. 1. The sugar is ribose in the case of RNA and 2′-deoxyribose in the case of DNA. The electronic transitions of the ether and hydroxyl groups of these saturated sugars begin at 200 nm, but their weak intensity is buried under the strong intensity of the electronic transitions of the aromatic bases. Electronic transitions of the phosphate group begin even further into the vacuum UV. Therefore, the CD of the nucleic acids that corresponds to the electronic transitions results from the bases. Open image in new window Figure 1 The electronic structure of the nucleic acid bases: adenine (A), guanine (G), cytosine (C) thymine (T), and uracil (U). ○, π electrons; ●, nonbonding electrons.

Protein secondary structure from circular dichroism spectra

Circular dichroism spectra of proteins are extremely sensitive to secondary structure. Nevertheless, circular dichroism spectra should not be analyzed for protein secondary structure unless they are measured to at least 184 nm. Even if all the various types ofβ-turns are lumped together, there are at least 5 different types of secondary structure in a protein (α-helix, antiparallelβ-sheet, parallelβ-sheet,β-turn, and other structures not included in the first 4 categories). It is not possible to solve for these 5 parameters unless there are 5 equations. Singular value decomposition can be used to show that circular dichroism spectra of proteins measured to 200 nm contain only 2 pieces of information, while spectra measured to 190 nm contain about 4. Adding the constraint that the sum of secondary structures must equal 1 provides another piece of information, but even with this constraint, spectra measured to 190 nm simply do not analyze well for the 5 unknowns in secondary structure. Spectra measured to 184 nm do contain 5 pieces of information and we have used such spectra successfully to analyze a variety of proteins for their component secondary structures.

The Circular Dichroism of Carbohydrates

Publisher Summary This chapter discusses the use of circular dichroism (CD) spectroscopy in the analysis of carbohydrates. The CD spectroscopy measures the difference in absorption between left- and right-circularly polarized light by an asymmetric molecule. The spectrum results from the interaction among neighboring groups, and is thus extremely sensitive to the conformation of a molecule. Commercial instrumentation measures the CD of electronic absorption bands in the range of 1000 to 190 nm. Nucleic acids and proteins both have absorption bands in this region, and CD has been used extensively to study these molecules. Most sugars are transparent in this region and so they have been relatively neglected. However, the diverse biological activities of sugars undoubtedly depend on their conformations. Thus, improvements in CD instrumentation for the short-wavelength region have stimulated interest in using this powerful technique for investigating the stereochemistry of sugar monomers, the configuration of intersaccharide linkages, the secondary structure of the polymers, and the interaction of sugars with themselves and with other biological molecules. This chapter details the ways in which modern CD instrumentation has been used to solve structural problems involving sugars. The discussion is limited to substituted and unsubstituted pyranoses and does not cover complexes that can be formed with various chromophores.