Biopolym. Cell. 2000; 16(5):369-379.
Structure and Function of Biopolymers
Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures
1Filenko A. M.
  1. Petr Bogach Institute of Physiology
    Taras Shevchenko National University of Kyiv
    2, Academika Glushkova Ave Str., Kyiv, Ukraine, 03187


Modern multi-angle light scattering, fast protein liquid chromatography and laser correlation spectroscopy used together give rather complete information about the distribution of different protein particles in solution and their characteristics. The data received by these methods on smooth muscle myosin light chain kinase (MLCK) as the object of investigation suggest that MLCK exists in solution as a mixture of oligomeric, dimeric and monomeric particles which contents at ionic strength close to physiological constitute 2, 53 and 45 wt. % correspondingly. An important point is that supramolecular kinase species content in eluate from a gel filtration column was much higher than their content at equilibrium. The contributions of oligomer, dimer and monomer in eluate at the exit from the column were 5.3, 81.5 and 13.2 wt. % accordingly. All three kinase species are characterized by prolonged lifetime. The transition from pure dimer into equilibrium state lasts for about 10 min. The kinase dimer is a rod-like structure with molecular mass of about 2-10 kDa and root mean square (RMS) radius Rt 22 nm. Oligomer is characterized by RMS radius Rs 80 nm. Its structure may be presented as a helical ring containing JO kinase molecules per turn with a number of turns about 10. Another more realistic explanation of the data obtained involves a rod-like or elongated spiral model according to which 6 kinase molecules, arranged in line or elongated spiral, form one structural unit, which must be a real oligomer (hexamer). About 17 such structural units, associated in parallel, form aggregates with molecular mass of about 101 kDa. Kinase spiral hexamer fits well the structure of smooth myosin filament with which the kinase is in close contact in vivo. Preliminary experiments with a number of other proteins (myosin, myosin subfragment 1, bovine serum albumin, chemotrypsin, papain) showed that all of them form supramolecular structures with prolonged time of transition from pure species to equilibrium distribution of monomers and supramolecular structures.


[1] Schulz GE, Schirmer RH. Principles of protein structure. New-York: Springer. 1979; 316 p.
[2] Mayr GW, Heilmeyer LM Jr. Skeletal muscle myosin light chain kinase. A refined structural model. FEBS Lett. 1983;157(2):225-31.
[3] Mayr GW. Interaction of calmodulin with muscle phosphofructokinase. Changes of the aggregation state, conformation and catalytic activity of the enzyme. Eur J Biochem. 1984;143(3):513-20.
[4] Mayr GW. Interaction of calmodulin with muscle phosphofructokinase. Interplay with metabolic effectors of the enzyme under physiological conditions. Eur J Biochem. 1984;143(3):521-9.
[5] Kosk-Kosicka D, Bzdega T, Wawrzynow A, Scaillet S, Nemcek K, Johnson JD. Erythrocyte Ca2+-ATPase: activation by enzyme oligomerization versus by calmodulin. Calcium Binding Proteins in Normal and Transformed Cells. Eds R. Pochet, D. Eric, M. Lawson, C. W. Heizmann. New York: Plenum Publ. Cor., 1990: 169-74.
[6] Flapper W, van den Oetelaar PJ, Breed CP, Steenbergen J, Hoenders HJ. Detection of serum proteins by high-pressure gel-permeation chromatography with low-angle laser light scattering, compared with analytical ultracentrifugation. Clin Chem. 1986;32(2):363-7.
[7] Hayashi Y, Matsui H, Takagi T. Membrane protein molecular weight determined by low-angle laser light-scattering photometry coupled with high-performance gel chromatography. Methods Enzymol. 1989;172:514-28.
[8] Kijima Y, Takagi T, Shigekawa M, Tada M. Protein-protein interaction of detergent-solubilized Ca2(+)-ATPase during ATP hydrolysis analyzed by low-angle laser light scattering photometry coupled with high-performance gel chromatography. Biochim Biophys Acta. 1990;1041(1):1-8.
[9] Rarity JG, Owens PC, Atkinson T, Seabrook RN, Carr RJ. Light-scattering studies of protein association. Biochem Soc Trans. 1991;19(2):489-90.
[10] Wen J, Arakawa T, Philo JS. Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal Biochem. 1996;240(2):155-66.
[11] Kunitani M, Wolfe S, Rana S, Apicella C, Levi V, Dollinger G. Classical light scattering quantitation of protein aggregates: off-line spectroscopy versus HPLC detection. J Pharm Biomed Anal. 1997;16(4):573-86.
[12] Wyat PJ. Calcium transients and resting levels in isolated smooth muscle cells monitored with quin 1 Anal chim acta. 1993; 272(3):1-40.
[13] Sobieszek A, Barylko B. Enzymes regulating myosin phosВ­phorylation in vertebrate smooth muscle. Smooth Muscle Contraction. Ed. N. L. Stephens. New York: Marcel Dekker, 1984: 283-316.
[14] Sobieszek A. Regulation of smooth muscle myosin light chain kinase. Allosteric effects and co-operative activation by calmodulin. J Mol Biol. 1991;220(4):947-57.
[15] Sobieszek A, Strobl A, Ortner B, Babiychuk EB. Ca(2+)-calmodulin-dependent modification of smooth-muscle myosin light-chain kinase leading to its co-operative activation by calmodulin. Biochem J. 1993;295 ( Pt 2):405-11.
[16] Adelstein RS, Klee CB. Purification and characterization of smooth muscle myosin light chain kinase. J Biol Chem. 1981;256(14):7501-9.
[17] Braginskaya TG, Dobichin PD, lvanova MA. Analysis of polydispersity by photon correlation spectroscopy. Phys Scripta. 1983; 26(5):309-15.
[18] Nicoli DF, McKenzie DC, Wu JS. Application of dynamic light scattering to particle size analysis of macromolecules. Int Lab. 1992; 24(8):32-7.
[19] Ausio J, Malencik DA, Anderson SR. Analytical sedimentation studies of turkey gizzard myosin light chain kinase and telokin. Biophys J. 1992;61(6):1656-63.
[20] Lowey S, Goldstein L, Cohen C, Luck SM. Proteolytic degradation of myosin and the meromyosins by a water-insoluble polyanionic derivative of trypsin: properties of a helical subunit isolated from heavy meromyosin. J Mol Biol. 1967;23(3):287-304.
[21] Carlson FD, Fraser AB. Intensity fluctuation autocorrelation studies of the dynamics of muscular contraction. Photon Correlation and Light Beating spectroscopy. Eds H. Z. Cummins, E. R. Pike. New York: Plenum press, 1974: 519-32.
[22] Privalov PL. Stability of proteins. Proteins which do not present a single cooperative system. Adv Protein Chem. 1982;35:1-104.
[23] Olson NJ, Pearson RB, Needleman DS, Hurwitz MY, Kemp BE, Means AR. Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc Natl Acad Sci U S A. 1990;87(6):2284-8.
[24] Filenko AM, Danilova VM, Sobieszek A. Smooth muscle myosin light chain kinase, supramolecular organization, modulation of activity, and related conformational changes. Biophys J. 1997;73(3):1593-606.
[25] Sobieszek A. Vertebrate smooth muscle myosin: enzymatic and structural properties. The Biochemistry of Smooth Muscle. Ed. N. L. Stephens. Baltimore: Univ. Park press, 1977: 413-443.
[26] Sobieszek A. Cross-bridges on self-assembled smooth muscle myosin filaments. J Mol Biol. 1972;70(3):741-4.