William D. McCubbin

Circular dichroism studies on concanavalin A

Abstract Optical rotatory dispersion and circular dichroism studies on native and demetallized concanavalin A indicate that this protein consists essentially of a mixture of β-form and random coil. The induction of α-helix in the native protein is accomplished through the use of 2-chloroethanol. The sequential addition of transition metal, Ca2+ and α-methyl-D-glucopyranoside to solutions of demetallized protein results in small but reproducible alterations in the aromatic CD spectrum, the largest changes being noted with calcium addition. Aromatic CD difference spectral studies suggest that a single transition may be involved in the binding of metal and α-MG to the protein; the wavelength position of this transition implicates tyrosine residues. At the same time, no significant variations occur in the secondary structure of the protein as reflected by the constancy of the far ultraviolet CD spectrum with ligand complexation. Exploration of the CD aromatic fine structure as a function of pH indicates that both tyrosine and tryptophan chromophores are involved.

Molecular weight studies on concanavalin A.

Summary Molecular weight studies have been performed on solutions of Concanavalin A (Con A) at pH values of 5.2, 7.0 and 7.5. The results suggest that Con A exists as a mixture of a species with molecular weight of 30,000 g/mole and higher aggregates. At pH 5.2 dimers predominate resulting in whole cell weight average molecular weights of approximately 55,000 g/mole, while at neutral pH, trimers are indicated, yielding molecular weights of the order of 90,000 g/mole. Electrophoresis on sodium dodecyl sulfate polycrylamide gels showed the presence of one major band with molecular weight 30,000 – 35,000 g/mole and two minor ones with M values of 13,000 – 15,000 and 21,000 – 23,000 g/mole, respectively. Equilibrium centrifugation of maleyl Con A, and Con A in 5 M guanidine HCl yielded a subunit molecular weight close to 17,000 g/mole. The two species plot of Roark and Yphantis suggested a species of molecular weight 13,000 – 15,000 g/mole in these media. If the preparative molecular weight of Con A is assumed to be around 70,000 g/mole then our results suggest that the molecule can exist as “half-mers” and under appropriate conditions, these can further dissociate into the basic subunit of ≈17,000 g/mole. A logical inference of these findings is that Con A is apparently composed of four subunits.

Purification and characterization of Aspergillus niger exo-1,4-glucosidase

A specific exo-1,4-glucosidase (1,4-alpha-D-glucan glucohydrooase, EC 3.2.1.3) from Aspergillus niger has been partially purified and subsequently characterized by biochemical, physico-chemical and optical methods. Molecular sieve chromatography yields an enzyme with maximal activity at pH 4.2-4.5 close to its isoelectric point. Reduction and carboxymethylation leads to complete loss of activity and O-acetylation of 3 of the 13 tyrosine residues results in loss of 20 % of the activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that the native enzyme consists of two major components of molecular weights 63 000 and 57 500, respectively. Small amounts of dissociated material of molecular weight 28 000 and 16 000 as well as aggregates of the order of 100 000 are also present to the extent of 2-5% of the total potein. Following reduction and carboxymethylation under forcing conditions, the bands around 60 000 diminish and the 28 000-30 000, 16 000 and aggregate bands are dominant...

Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila: Biochemical characterization of the major structural domain

The tetragonally arranged S-layer of Aeromonas hydrophila contains two morphological domains. The mature S-layer protein of A. hydrophila has a subunit molecular weight of 52,000, and has been reported to contain two structural domains. Here a mutant has been isolated which produces an S-layer of subunit molecular weight 38,650 as determined by sedimentation analysis. This truncated S-protein was exported via the periplasm to the cell surface, but could not self-assemble into a tetragonal array or be anchored to the cell surface. Instead the truncated protein formed cup-like structures which were purified and characterized biochemically. Automated Edman degradation showed that the truncated protein comprised the amino-terminal structural domain of the S-protein. This domain had an increased hydrophobic amino acid content relative to the wild-type protein, and contained approximately 42% beta-sheet, 10% alpha-helix, and 19% beta-turn. Differences in alpha-helix and beta-turn contents between the wild-type and truncated proteins were observed when the effects of pH and SDS were examined, indicating that the carboxy terminus influences the effects of environmental change on the conformation of the S-protein. This lesser carboxy-terminal array also appears to be required for both correct array morphology, and array anchoring, while the greater amino-terminal domain appears to comprise the major morphological core of the surface array.

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

Hydrodynamic properties of bovine cardiac troponin.

A refined knowledge of muscle regulation at the level of the thin filaments can be obtained from a hydrodynamic study of the troponin complex and its subunits. This approach has already been used to determine the nature of the Caz’-induced conformational change in skeletal TN-C* [ 1,2] and its interaction with other subunits [2,3]. However, little or no work has been reported on the hydrodynamic properties of intact ‘native’ troponin, probably due to the difficulties in achieving clean preparations without resorting to denaturing conditions. Preparative methodology has advanced to the point where such studies can now be carried out for both skeletal and cardiac troponin. Lovell and Winzor [4] have demonstrated that ox skeletal troponin undergoes a concentration-dependent associationdissociation equilibrium which is also affected by pH, ionic strength and temperature, In this study, bovine cardiac troponin has been examined by sedimentation velocity, sedimentation equilibrium and analytical gel filtration. The results indicate that cardiac troponin also aggregates as a function of protein concentration, although in this case there is little evidence for dissociation into subunits. Gel filtration studies, extrapolated to low protein concentration, suggest that the troponin complex may be asymmetric in solution. There appears to be no significant effect of

The effect of terbium on the structure of actin and myosin subfragment 1 as measured by circular dichroism

Substitution of Ca2+ by La3+ or Tb3* produced a pronounced inhibitory effect on the ATPase activity of myosin S-l alone, or in combination with F-actin [ 11. To gain further insight into the possible mechanism of action of En3” a CD investigation was carried out, wherein the effect of Tb3+ on the secondary structure of Gand F-actin, myosin S-l, as well as comb~at~ons of these proteins, was established. The results suggest that Tb3’ induces structural decreases in all these proteins and interactions between myosin S-l and actin seem to be weakened in the presence of this lanthanide.

Circular dichroism and fluorescence studies on troponin—tropomyosin interactions

The regulatory apparatus for vertebrate striated muscle contraction is located at the thin filaments and consists of troponin (a complex of three separate subunits) in interaction with tropomyosin and actin [l-3]. It has recently been shown that troponin, tropomyosin and actin are present in a 1: 1:7 molar ratio in myofibrlls [4]. In 1963, Lowy and Hanson suggested that tropomyosin particles were longitudinally arranged in the two long pitch grooves of the F actin double helix [5], and this has presently been confirmed by several laboratories utilizing X-ray and electron diffraction techniques [6-81. These studies also demonstrated that the precise location of tropomyosin on the actin helix depends on the state of troponin and the level of calcium ions present. In the relaxed state, i.e., at low levels of calcium, tropomyosin molecules are in such a position as to prevent the interaction of the myosin ‘heads’ with actin. The binding of calcium ions by troponln C produces a dramatic conformational change in this molecule [9] which apparently is relayed via troponin T to tropomyosin, and this entity moves nearer the middle of the actin groove, thereby permitting the interaction

Equilibrium dialysis binding studies of 1,N6-ethenoadenosine diphosphate (εADP) to myosin, heavy meromyosin, and subfragment one

Abstract The binding of the fluorescent analog of adenosine diphosphate (ADP) ∗ , 1,N 6 -ethenoadenosine diphosphate (eADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for eADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 ( ± .5) × 10 −5 M −1 . This is identical to the values found by Young (J. Biol. Chem., 242 , 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of eATP versus ATP by S1 were studied. Values of V max and K m were 25 μM phosphate sec −1 (gm protein) −1 and 5 × 10 −5 M −1 for ATP, and 80 μN phosphate sec −1 (gm protein) −1 and 45 × 10 −5 M −1 for eATP. The results indicate that the increased V max that occurs when eATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of eATP versus ADP. This in turn suggests that the increase in V max may be due to an increased hydrolytic rate of eATP vs ATP in the enzyme substrate complex.

Comparative calcium binding and conformational studies of turkey and rabbit skeletal troponin C

Troponin C from turkey skeletal muscle has been compared with its chicken counterpart in terms of amino acid composition and fragmentation patterns and with rabbit TN‐C by Ca2+ binding and conformational response to Ca2+ as monitored by CD and fluorescence. Cyanogen bromide and tryptic digestion mixtures of chicken and turkey TN‐C have been separated by reversed‐phase HPLC. The similarity of the elution profiles, along with the almost identical amino acid compositional data, suggest that the sequences are essentially equivalent. Both turkey and rabbit TN‐C bound 2 mol Ca2+/mol protein at pH 5.3, while at pH 6.8, this figure was raised to 4 mol/mol protein. Circular dichroism and fluorescence measurements indicated that the conformations of the two proteins responded in a very similar manner to the presence of Ca2+