William F. Harrington

A model for the myosin molecule

Abstract 1. The molecular weight of myosin has been determined in 0.5 M KCl and 5 M guanidine · HCl. 2. 1. The molecular weight, using the Archibald approach to equilibrium method is strongly dependent on protein concentration in both solvents giving infinite dilution values of 6.19·10 5 in 0.5 M KCl and 2.06·10 5 in 5 M guanidine·HCl. 3. 2. Sedimentation, diffusion and viscosity studies on myosin in 5 M guanidine·HCl provide estimates of the molecular weight in reasonable agreement with the Archibald studies. 4. 3. Preferential binding of guanidine·HCl by myosin has been determined and shown to be of the order of 5% or less. 5. 4. Utilizing these molecular weight determinations and available X-ray diffraction, light scattering and optical rotatory data, a model for the myosin molecule is proposed, based on three equal weight polypeptide chains in the form of a three-stranded α rope.

The structure of collagen and gelatin.

Publisher Summary This chapter reviews that collagen constitutes the major protein component of skin, bone, tendon, and all the other forms of connective tissue. An understanding of collagen seems to the clinician to be a necessary prerequisite to a rational attack on many and diverse connective tissue disorders currently lumped together as “collagen diseases.” The unusual amino acid composition of collagen had also been recognized for some time. One-third of the residues of all collagens seemed to be glycine, while about one-fourth were proline and hydroxyproline. However, the stereochemical consequences of the presence of these residues has only become clear as a result of the detailed studies of synthetic homo- and copolymers of glycine and proline. Consideration of such synthetic polypeptides as simplified models of certain features of collagen and gelatin has been extremely helpful in recent years, and constitutes the rationale for the inclusion of a section dealing specifically with these synthetic polypeptides in this chapter. It also reviews that the collagen ⇆ gelatin transformation in solution has been recognized as a reversible first-order phase transition, subject to the same physical laws which govern the crystalline ⇆ amorphous phase transitions observed in systems of linear polymers. The direct relationship between the transition in solution and the well-known thermal shrinkage phenomenon exhibited by collagen fibers has also been established.

Role of pyrrolidine residues in the structure and stabilization of collagen

The physical properties of two neutral salt-soluble invertebrate collagens of markedly different total imino acid and hydroxyproline content have been compared. Measurements of sedimentation velocity, viscosity and optical rotation indicate that both Ascaris cuticle and earthworm cuticle collagens behave as rigid, asymmetric, rod-like particles in solution, with component polypeptide chains in the helical configuration of poly- L -proline II type and with molecular weights appreciably greater than those reported for the vertebrate tropo-collagens. Ascaris collagen (29% Pro; 2%Hypro) undergoes a thermal transition (collagen → gelatin) at 52°C (T D ) whereas earthworm collagen (0·8% Pro; 17% Hypro) exhibits a T D of 22°C. When these results are considered with the thermal denaturation temperatures of other soluble collagens of known composition, it is concluded that the total pyrrolidine (Pro + Hypro) rather than the Hypro content alone, is the significant feature in the stabilization of the collagen structure. The distribution of pyrrolidine residues in various collagens has been estimated statistically and leads to the conclusion that triplets containing neighboring pyrrolidine residues are thermally stable. The re-naturation rates of Ascaris and earthworm gelatins have been measured at low temperature and their relationship to the pyrrolidine content and distribution considered.

The correlation of ribonuclease activity with specific aspects of tertiary structure

Various physical parameters of bovine pancreatic ribonuclease and of some of its derivatives prepared by oxidation or reduction of disulfide bridges, methylation, and proteolytic digestion, have been investigated. Ultraviolet spectrophotometric measurements appear to establish a correlation between specific spectral properties of the materials and their enzymic activity. Viscometric and optical rotary studies, on the other hand, indicate that minor modifications in secondary structure may occur without detectable inactivation. Polyvalent anions and polyanions almost completely prevent the unfolding and spectral shift effect of 8 M urea, and of guanidium ions at concentrations between 1 and 3 M. These findings suggest that the full activity of ribonuclease, in the presence of such denaturing agents, is due to a refolding of the protein under the influence of ribonucleic acid.

Subunit structure of glyceraldehyde-3-phosphate dehydrogenase

The molecular weight of glyceraldehyde-3-phosphate dehydrogenase isolated from both pig and rabbit muscle has been determined from short-column sedimentation equilibrium experiments, giving 145,000 ± 6000 for both species. Velocity sedimentation, viscosity and sedimentation equilibrium studies of the enzyme in aqueous 5 M -guanidine-HCl solutions reveal that in this solvent glyceraldehyde-3-phosphate dehydrogenase is dissociated into monodisperse sub-units of molecular weight 36,300 ± 1500.

Evidence for structural changes in vertebrate thick filaments induced by calcium.

Abstract Paired sedimentation studies of isolated, native thick filaments at pH 6.8, I = 0.12 and in the presence of 0.3 m m -free Mg 2+ show that the sedimentation coefficient increases with Ca 2+ concentration (pCa † midpoint = 5.5), leveling off at pCa 4.7. The addition of ATP or ADP (5 m m ) has no effect on the hydrodynamic changes induced by Ca 2+ . At much higher free Mg 2+ concentrations (5 m m ), the midpoint of the transition is shifted to pCa = 5.3. Viscosity measurements of the filament system under comparable conditions reveal a decrease in the relative viscosity over the same range of Ca 2+ concentration. Synthetic filaments prepared from purified myosin free of C-protein also show the same behavior. Native filaments from which myosin heads have been removed by treatment with papain do not show Ca 2+ dependence. The dependence of the sedimentation coefficient of filament on protein concentration, as measured by differential sedimentation, is unaffected by Ca 2+ , indicating that the changes in hydrodynamic properties are probably not related to aggregation of the filaments. The Ca 2+ effects are reversible and are not observed on replacing Ca 2+ by Mg 2+ . Binding studies carried out at low ionic strength reveal two binding sites for Ca 2+ ( K a = 1.7 × 10 5 m −1 ) per mole myosin within the filament and evidence is presented showing that the DTNB light chain is the site of binding. The combined results are interpreted as indicating that thick filaments of vertebrate muscle undergo conformational changes at physiological levels of Ca 2+ and provide evidence for a Ca 2+ -sensitive regulatory mechanism at the level of the thick filament.

Studies on the structure of poly-l-proline in solution

Abstract 1. 1. The specific rotation of poly- l -proline II ([ α ] D 25 = − 540°) in water has been estimated making use of the theory of Fitts and Kirkwood for the optical rotation of helical molecules and the reported specific rotations of glycine-proline copolymers. 2. 2. The optical rotation, temperature dependence of rotation and rotatary dispersion of polyproline II in various solvents is reported. In those solvents of low water activity, the specific rotation changes to a limiting value of around — 250° while sedimentation and viscosity studies demonstrate a marked decrease in the asymmetry of the molecule. 3. 3. The mutarotation of poly- l -proline I ([ α ] D = + 40 °) in water has been followed by rotatory dispersion, viscosity and sedimentation studies. 4. 4. The above results are shown to be consistent with the proposal that polyproline I exists in aqueous solution as a right-handed helix with peptide bonds in the cis -configuration whereas polyproline II exists in aqueous solutions as a left-handed helix with peptide bonds in the trans -configuration.

Formation and stabilization of the collagen-fold

Abstract The mutarotation of ichthyocol gelatin at low temperatures has been examined over a wide range of protein concentrations in the region below the gelation level. The rates of mutarotation as well as the final values of specific rotation are shown to be independent of concentration suggesting that the configurational changes responsible for rotation depend on intramolecular events. Some type of hydrogen bonding appears to be involved in the formation and stabilization of the collagen-fold since no mutarotation is observed in concentrated aqueous solutions of urea or guanidine-HCl. Moreover, the melting profiles of chilled gelatin solutions in H 2 O and D 2 O reveal a significant increase in melting temperature (3.7 °C.) in D 2 O over that in H 2 O. Rate measurements of mutarotation reveal that (1) the process obeys second-order kinetics with respect to the amount of gelatin in the unfolded configuration; and (2) the rate of mutarotation at a fixed low temperature is much greater in D 2 O than in H 2 O. A mechanism for the formation of the collagen-fold is proposed in which water molecules act to stabilize the collagen-fold configuration through formation of doubly hydrogen-bonded bridges between adjacent carbonyl groups. The kinetics are interpreted in terms of an intramolecular crystallization process originating from preformed nuclei.

Studies on the tryptic digestion of myosin

Abstract The digestion of myosin by trypsin has been followed by measuring: (1) the uptake of base in the pH-stat, (2) changes in viscosity, (3) non-protein N formation, (4) changes in sedimentation properties and (5) changes in optical rotation. The pH-stat studies point to the existence of 2 parallel first-order reaction classes with markedly different velocity constants. Approximately 64 peptide bonds are cleaved in the fast reaction and 240 peptide bonds in slow reaction. The kinetic data obtained by means of the different physicochemical methods are consistent with a model of myosin in which a small number polypeptide chains are arranged parallel to one another throughout the lenght of the myosin rod. Some segments of this rod structure are highly ordered and some are more randomly disposed. The enzyme attacks the random regions at a much higher rate than the folded areas. Thus the early appearance of large intermediates in proteolysis would appear to be a reflection of the difference in the rates of attack of these different segments of the molecule, and does not imply the pre-existence of any sort of subunit structure.

Enzymic studies of the gelatin → collagen-fold transition☆

Abstract The molecular configuration of ichthyocol gelatin has been examined at various temperatures, using the proteolytic enzyme collagenase as a structural probe. The kinetics of proteolysis were followed by a combination of pH-stat and optical rotation methods. Above 27°, where ichtyocol gelatin exists in solution as a single-chain, randomly-coiled molecule, proteolysis follows simple first-order kinetics as measured by both of the above methods. On lowering the temperature, the kinetics become more complex, proteolysis now following the sum of two parallel first-order reactions proceeding at markedly different rates. On the basis of these studies (and supporting optical rotation, viscosity and light-scattering experiments on temperature and dilution effects), the following model for the gelatin → collagen-fold transition emerges: (a) the primary step appears to be a local intra-chain configurational change involving the locking of proline residues into the poly- L -proline II configuration; (b) this is a necessary precursor of, and leads directly to, the development of a loose, poly- L -proline II-type helix along the gelatin molecules; which (c) then permits the close packing required for inter-chain associations.