N-acetylserine in horse muscle acylphosphatase.

A ninhydrin-negative peptide fraction obtained from tryptic digest of carboxymethyl acylphosphatase was isolated by chromatography on a column of PA 28 Beckman resin and analysed for the amino acid composition. Degradation with carboxypeptidase B and A indicated that the sequence of this peptide was: X-Thr-Ala-Arg. The amino-terminal residue was identified as N-acetylserine by high voltage electrophoresis. It is therefore suggested that the sequence of the NH2-terminal portion of CM-acylphosphatase is N-acetyl-Ser-Thr-Ala-Arg. Digestion with carboxypeptidase A and B indicated also that the COOH-terminal portion of CM-acylphosphatase is-Arg-Tyr-OH.     

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.     

Predicted secondary structure of bovine prothrombin fragment 1 and related proteins in different environments by circular dichroism spectroscopy.

Circular dichroism spectroscopy was used to investigate the structure of bovine prothrombin fragment 1 (BF1) and related proteins in several environments. The conformational change induced in BF1 by the addition of Mg[II] ions was found to be different from that induced by Ca[II] or Sr[II]. The Ca[II] and Sr[II] conformations appear to differ only slightly from the apo-metal conformation. The conformation of the 1-45 fragment of prothrombin, however, is markedly different than the conformation of the same fragment in the presence of either Ca[II] of Mg[II]; both of the latter structures differ substantially from one another. The presence of phospholipids has almost no effect on the structure of either BF1 or the 1-45 fragment; in the presence of both phospholipids and Ca[II] a structural change is seen for the 1-45 fragment but not BF1 (relative to the protein alone). The addition of phospholipids to the Mg[II]/BF1 structure did not induce a CD-detectable conformational change, while the addition of phospholipids to the Ca[II]/BF1 or Sr[II]/BF1 structures induced a change to a conformation similar in secondary structure composition to the relative apometal structures.     

Inhibition of horse muscle acylphosphatase by pyridoxal 5'-phosphate.

It has been shown that horse muscle acylphosphatase is inhibited by pyridoxal 5'-phosphate and that the inhibition is pH dependent, reversible and competitive with respect to substrate binding. Spectral analysis on the EI complex demonstrates the presence of a Schiff base. Reduction of the pyridoxal 5'-phosphate-inhibited enzyme with sodium borohydride, followed by amino acid analysis, produces a diminution of the free lysine peak and the appearance of a new peak corresponding to epsilon-pyridoxyllysine. The results suggest that there is at least one NH2-lysyl residue of horse muscle acylphosphatase at or near the active site of the enzyme.     

Stability and kinetic behavior of carboxymethylated horse muscle acylphosphatase.

Horse muscle acylphosphatase consists of a main chain S-S bound to glutathione. It was found that removal of the glutathione by reduction and successive carboxymethylation of the only cysteine of the main chain affects the stability of the enzyme, mainly with respect to thermal inactivation. On the other hand, the kinetic properties of the enzyme are affected very little.     

The optimization of protein secondary structure determination with infrared and circular dichroism spectra.

We have used the circular dichroism and infrared spectra of a specially designed 50 protein database [Oberg, K.A., Ruysschaert, J.M. & Goormaghtigh, E. (2003) Protein Sci. 12, 2015-2031] in order to optimize the accuracy of spectroscopic protein secondary structure determination using multivariate statistical analysis methods. The results demonstrate that when the proteins are carefully selected for the diversity in their structure, no smaller subset of the database contains the necessary information to describe the entire set. One conclusion of the paper is therefore that large protein databases, observing stringent selection criteria, are necessary for the prediction of unknown proteins. A second important conclusion is that only the comparison of analyses run on circular dichroism and infrared spectra independently is able to identify failed solutions in the absence of known structure. Interestingly, it was also found in the course of this study that the amide II band has high information content and could be used alone for secondary structure prediction in place of amide I.     

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 < 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.     

Stability of horse muscle acylphosphatase to heat and to urea.

The thermal stability of horse muscle acylphosphatase was investigated by measuring the inactivation constants at various pH and temperature values, and by differential spectra technique. This enzyme has high thermal stability in an acidic environment but is inactivated in an alkaline medium. It was found that the enzyme can be protected against such inactivation at pH 8.0 by increasing its concentration and the ionic strength of the solution. The effect of high urea concentrations on stability was also measured. It was found that spectral changes at 230 nm are related to urea inactivation of the enzyme, and that the enzymatic activity can be instantly and almost completely restored by dilution of the urea.     

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy is a fast, powerful, well-established, and widely used analytical technique in the biophysical and structural biology community to study protein secondary structure and to track changes in protein conformation in different environments. The use of the intense light of a synchrotron beam as the light source for collecting CD measurements has emerged as an enhanced method, known as synchrotron radiation circular dichroism (SRCD) spectroscopy, that has several advantages over the conventional CD method, including a significant spectral range extension for data collection, deeper access to the lower limit (cut-off) of conventional CD spectroscopy, an improved signal-to-noise ratio to increase accuracy in the measurements, and the possibility to collect measurements in highly absorbing solutions. In this review, we discuss different applications of the SRCD technique by researchers from Latin America. In this context, we specifically look at the use of this method for examining the secondary structure and conformational behavior of proteins belonging to the four main classes of the hierarchical protein domain classification CATH (Class, Architecture, Topology, Homology) database, focusing on the advantages and improvements associated with SRCD spectroscopy in terms of characterizing proteins composed of different structural elements.     

The complete amino acid sequence of horse muscle acylphosphatase

The amino acid sequence of horse muscle acylphosphatase is given in the present paper. The carboxymethylated enzyme consists of a single polypeptide chain of 98 amino acid residues with an acetyl group blocking the NH2 terminus and a tyrosine at the COOH terminus. The calculated molecular weight of the native protein, a mixed disulfide with glutathione, is 11,365. The carboxymethylated protein was cleaved by cyanogen bromide. The three expected fragments were purified; moreover, an additional fragment, derived from a partial failure of cleavage at methionine-24, was purified and characterized. The structures of the cyanogen bromide fragments were established by subfragmentation with endopeptidases, and the sequences of the overlapping subfragments were determined. From the results, it was possible to order the peptides within the sequence and then to establish the complete primary structure of the enzyme.     

Purification of horse muscle acylphosphatase antibodies by affinity chromatography

Horse muscle acylphosphatase antibodies were obtained by immunizing rabbits with the highly purified antigen cross-linked with glutaraldehyde. Specific antibodies were purified from the immunoglobulin fraction by affinity chromatography using a matrix coupled with the pure antigen as immunoadsorbent. The purified antibodies were partially characterized by immunodiffusion and immunoprecipitin techniques. These antibodies could be used to study aspects of the muscle acylphosphatase structure, localization and other biological properties.     

Determination of secondary structure of globular proteins using circular dichroism spectra

A ridge regression method is presented for prediction of the secondary structure of proteins by the circular dichroism spectra (CD) from 190 to 236 nm. Eight types of the secondary structure were calculated on a microcalculator. The method is based on the X-ray data of Kabsh and Sander. The teaching rule is constructed on CD spectra of 30 proteins of all structural classes of the globular proteins (alpha, alpha/beta, alpha + beta and beta-proteins). The errors of the methods are analysed by removing each protein from the reference set and analyzing its structure in terms of the remaining proteins. Correlation coefficients and root-mean square deviations between CD and X-ray data were: 0.99 and 0.03 for alpha-helix, 0.86 and 0.02 for 3(10)-helix, 0.92 and 0.06 for antiparallel beta-sheet, 0.86 and 0.03 for parallel beta-sheet, 0.94 and 0.01 for T3 beta-turn, 0.85 and 0.02 for other beta-turn, 0.84 and 0.03 for S-bends, 0.83 and 0.04 for "random" structure.     

Determination of protein secondary structure from circular dichroism spectra. III. Protein-derived base spectra of circular dichroism for antiparallel and parallel beta-structures

Protein-derived basic CD spectra for alpha-helix, antiparallel and parallel beta-structures, beta-bends and irregular form of proteins have been determined from the experimental CD spectra of six (myoglobin, lysozyme, ribonuclease A, papain, lactate dehydrogenase, subtilisin BPN') or seven (glyceraldehyde-3-phosphate dehydrogenase added) reference proteins and the analysis of the X-ray data. The secondary structures of thirteen proteins (seven reference and six additional ones) have been analysed using the basic CD spectra thus obtained. The data obtained have been compared with the results of the X-ray data analysis. It is shown that the accuracy of determination of the beta-structure and beta-bends contents using our basic CD spectra is about 2-3 times better than using the basic spectra reported by Chang et al. (Analyt. Biochem. 91, 13-31, 1978).     

Identification of three continuous antigenic sites in horse muscle acylphosphatase

The main antibody-combining sites of horse skeletal muscle acylphosphatase were mapped by preparing and purifying CNBr, tryptic and peptic peptides from the pure enzyme, and looking for the immunoreactivity of each peptide by the dot-immunobinding assay using specific polyclonal antienzyme antibodies previously purified by immunoaffinity chromatography. The immunoreactive peptides were identified on the basis of either their elution times in the fingerprint analysis or amino acid composition, or both, by comparison with the known enzyme amino acid sequence. All the CNBr as well as two tryptic and two peptic peptides were immunopositive, leading to identification of three main continuous antigenic sites on the enzyme molecule. The strong inhibition (92%) of the antigen-antibody reaction carried out in the presence of antibodies previously incubated with the immunoreactive peptide mixture supports the possibility that, at our experimental condition, the three identified antigenic domains contain the main antigenic determinants of the enzyme. The relationship between structure and antigenicity of the immunoreactive peptides is discussed in detail.     

Extended circular dichroism measurements using synchrotron radiation show that the assembly of clathrin coats requires no change in secondary structure.

A number of models have been proposed for the assembly of clathrin triskelia into coats. However, little is known of the effects of assembly on triskelion structure. A more detailed knowledge of the way in which assembly affects triskelion structure would be valuable for assessing the relative merits of the proposed models. The development of a vacuum-ultraviolet circular dichroism (CD) instrument that uses synchrotron radiation as a light source has allowed us to extend the range of CD measurements to shorter wavelengths. This has greatly increased signal quality even for highly scattering samples. Also, we have improved CD data analysis to provide standard deviations for calculated secondary structure content. These developments have increased the precision of CD analysis beyond what has been thus far possible. Using these developments, we have determined the secondary structure content of all components of coat protein, under both assembly and dissociating conditions. The assembly of coats does not incur any change in secondary structure content, but a 10% loss of triskelion helical content accompanies assembly in the absence of AP-2. We conclude that coat assembly requires no detectable reorganization of triskelion structure. Our result indicates that AP-2 stabilizes helical structure in the triskelion, and we propose that this increases triskelion rigidity, restricting the range of coat sizes.