Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures
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 m...
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Інститут молекулярної біології і генетики НАН України
2000
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| Цитувати: | Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures / A.M. Filenko // Биополимеры и клетка. — 2000. — Т. 16, № 5. — С. 369-379. — Бібліогр.: 26 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1528322025-02-23T17:27:02Z Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures Дослідження олігомерних форм білків методами світлорозсіювання: надмолекулярні структури кінази легких ланцюгів міозину гладеньких м'язів Исследование олигомерных форм белков методами светорассеяния: надмолекулярные структуры киназы легких цепей миозина гладких мышц Filenko, A.M. Структура и функции биополимеров 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. Сучасні методи багатокутового світлорозсіювання в поєднанні зі швидкісною хроматографією білків та лазерною кореляційною спектроскопією дають досить детальну інформацію щодо розподілу білкових частинок у розчині, їхнього розміру та молекулярної маси. Дані, отримані при дослідженні кінази легких ланцюгів міозину гладеньких м'язів, свідчать про те, що цей білок існує в розчині як рівноважна суміш олігомерних, дймерних та мономерних часток у кількісному співвідношенні 2, 53 та 45 вагових % відповідно. На виході з гель-фільтраційної колонки рівновага значно зсунута в бік олігомерних форм кінази і час переходу до рівноважного стану становить приблизно 10 хв. Димер кінази має стрижнсвидну структуру з середньоквадратичним радіусом (СКР) біля 22 нм. Для олігомеру СКР складає біля 80 нм. Його структуру можна представити у вигляді спірального кільця із 10 витків з 10 молекулами кінази на виток. Структуру олігомера добре описують також стрижневидна або спіралевидна моделі з шістьма молекулами, розміщеними вздовж лінії або витягну тої спіралі. Біля 17 таких шестимолекулярних елементів утворюють паралельно асоційовані агрегати. Попередні до слідження показали, що низка інших білків також існує в розчині як рівноважна суміш мономерів і надмолекулярних структур з тривалим часом життя. Современные методы многоуглового свсеторассеяния совместно со скоростной хроматографией белков и лазерной корреляционной спектрскопией дают достаточно полную информацию о распределении белковых частиц в растворе, их размере и молекулярной массе. Данные, полученные при исследовании киназы легких цепей миозина гладких мышц, свидетельствуют, что этот белок существует в растворе как равновесная смесь олигомерных, димерных и мономерных частиц в количественном соотношении 2, 53 и 45 весовых % соответственно. На выходе из гель-фильтрационной колонки равновесие сильно сдвинуто в сторону олигомерных форм киназы и время перехода в равновесное состояние составляет приблизительно 10 мин. Димер киназы имеет стержневидную структуру со среднеквадратичным радиусом (СКР) около 22 нм. Для олигомера СКР составляет около 80 нм. Его структуру можно представить в виде спирального кольца из 10 витков с 10 молекулами киназы на виток. Структуру олигомера хорошо описывают также стержневидная или спиралевидная модели с шестью молекулами, размещенными вдоль линии или вытянутой спирали. Около 17 таких шестимолекулярных элементов образуют параллельно ассоциированные агрегаты. Предварительные исследования показали, что ряд других белков также существует в растворе как равновесная смесь мономеров и надмолекулярных структур с продолжительным временем жизни. 2000 Article Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures / A.M. Filenko // Биополимеры и клетка. — 2000. — Т. 16, № 5. — С. 369-379. — Бібліогр.: 26 назв. — англ. 0233-7657 DOI:http://dx.doi.org/10.7124/bc.00057F https://nasplib.isofts.kiev.ua/handle/123456789/152832 591.175.4 en Биополимеры и клетка application/pdf Інститут молекулярної біології і генетики НАН України |
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Структура и функции биополимеров Структура и функции биополимеров |
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Структура и функции биополимеров Структура и функции биополимеров Filenko, A.M. Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures Биополимеры и клетка |
| description |
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. |
| format |
Article |
| author |
Filenko, A.M. |
| author_facet |
Filenko, A.M. |
| author_sort |
Filenko, A.M. |
| title |
Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures |
| title_short |
Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures |
| title_full |
Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures |
| title_fullStr |
Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures |
| title_full_unstemmed |
Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures |
| title_sort |
detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2000 |
| topic_facet |
Структура и функции биополимеров |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152832 |
| citation_txt |
Detection and characterization of protein oligomeric species by light scattering methods: myosin light chain kinase supramolecular structures / A.M. Filenko // Биополимеры и клетка. — 2000. — Т. 16, № 5. — С. 369-379. — Бібліогр.: 26 назв. — англ. |
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Биополимеры и клетка |
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I S S N 0233-7657. Биополимеры и клетка. 2000. Т. 16. № 5
Detection and characterization of protein oligomeric
species by light scattering methods: myosin light
chain kinase supramolecular structures
A. M. Filenko
Institute of Physiology, Taras Shevchenko Kiev University, Ukraine
Modem 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 2y 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, 8J.5 and
J 3.2 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-Ю5 kDa and root mean square (RMS) radius Rg 22 nm. Oligomer is
characterized by RMS radius Rg 80 nm. Its structure may be presented as a helical ring containing 10
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.
Introduction. Many proteins are known to function in
living cell as supramolecular structures, which include
2 and more molecules [1 ]. For instance the oligomer-
dependent modification of enzyme activity is well
demonstrated for such proteins as muscle phospho-
fructokinase [2—4 ] or erythrocyte Ca 2 +-ATPase [51.
The determination of real molecular weight and size
of different molecular species is of vital importance for
their characterization. At present there is no more
fundamental tool for measuring mass and size of
molecules than light scattering in conjunction with
liquid chromatography. This technique has been de
veloping intensively for last decades [6—12] and now
© A. M. FILENKO, 2000
complies with the most inexorable demands of many
researchers.
In this paper we demonstrate the possibilities of
this new approach for characterization of myosin light
chain kinase (MLCK), a key regulatory enzyme of
smooth muscle contractile system. We have also used
the laser correlation spectroscopy, which along with
multi-angle light scattering has enabled us to find out
equilibrium distribution of different kinase species in
weight percents. These two methods used together
permit to obtain rather complete characterization of
proteins studied.
In our investigations we have used for the first
time the procedure of decomposition of mass sensitive
and light scattering elution profiles into individual
monopeaks that allows more careful analysis of the
369
FILENKO A. M.
data obtained. We have also used for the first time
the interruption of elution in specified points to
evaluate time of different species transition to equilib
rium state.
Materials and Methods. Protein preparation.
MLCK and Ca2 +-calmodulin (CaM) were purified
from turkey gizzard as described elsewhere [13—15].
Their concentrations were determined using absorp
tion coefficient of Лш
1 % s s 1 1 . 4 and A21s °/o = 1.0,
respectively, for MLCK and CaM [16] . All light
scattering experiments were carried out in the buffer
AA of the following composition (mM): KC1, 60;
MgCl2, 2; dithioerythritol, 0.5; imidazole, 10; with pH
adjusted to 7.5 at 4 °С Unless otherwise stated
100 mM NaCl was added to this buffer during all
measurements. All other experimental details are
given in the corresponding legends to figures.
Laser correlation spectroscopy. Laser correlation
spectroscopy (LCS) was applied to detect directly
different MLCK species in solution. Helium-neon
laser («Spectrophysics», USA) operating in one mode
regime with wavelength 632.8 nm and power 50 mW
was used as the light source. Laser beam was focused
by precision optic system («Optimation GmbH», Ger
many). Emission scattered by the solution studied
was collected by the system of lenses at right angles
to the laser beam direction. To identify the kinase
particles some experiments were performed on light
scattering dependence on the angle of registration.
The apparatus was supplied with Goniometer
ALV/SP-86 («Optimation GmbH»). Autocorrelation
function of scattered light intensity fluctuations was
measured by correlator K7032 («Malvern, Instru
ments Ltd.», UK). Analysis of the autocorrelation
functions obtained was carried out on PC according to
the regulation procedure described elsewhere [17].
LCS together with the mathematical program of
regulation enables to obtain information about both
effective hydrodynamic diameters of particles and
their relative distribution in solution. The results of
sample measurements by the LCS method are presen
ted as a histogram, which reflects the form of the
particle size distribution function. The bar height in
the histogram is proportional to the relative contri
bution of the given size particles into the total light
scattering spectrum in a given interval. The regulation
program reconstructs the size distribution function in
32 points. Because of the broad range of the particle
sizes the distribution function is presented in the
logarithmic scale. To get rid of dust particles the
protein solution was cleared before measurement with
Millipore filter (0.22 /urn).
Multi-angle light scattering photometry. Multi-
angle laser light scattering in conjunction with the
system of fast protein liquid chromatography (FPLC)
was used to obtain characteristics of the MLCK
species (molecular mass and size) in the course of
their elution from chromatographic column. Light
scattering was measured on the Wyatt Dawn multi-
angle laser photometer, model F («Wyatt Technology
Corporation*, USA) at the laser wavelength 632.8 nm.
Signals from 18 photodiodes were sent to a 19 channel
A/D converter and then to a computer. The 19th
channel was used for a signal from a mass sensitive
UV detector placed between the column and flow cell
of the photometer. Standard base FPLC («Pharmacia
LKB Biotechnology*, Sweden) included the following
elements: controller GP-250, high precision pump
P-500, valve V-7 with 500 JU\ loop, recorder REC-102,
ultraviolet monitor UV-1. 8ml gel-filtration column
(6 x 284 mm) was packed with Sephacryl S-300. The
connecting tubing between the column and the photo
meter was selected of a possible minimal length so
that UV monitor — Dawn photometer volume delay
was 0.16 ml and column — UV monitor delay was
0.1 ml. Eluent flow rate was 0.25 ml/min. All solu
tions before using them for chromatography were
carefully degassed and filtered through Millipore
0.22 fim.
Light scattering data were analyzed on PC using
Wyatt ASTRA software. At calculations we assumed
values for specific refractive index increment dn/dc-
= 0.17 ml/g and for the second virial coefficient A2 =
= 0. The least-squares fit (LSF) degree was chosen 2
because of rather large MLCK oligomer size. Wyatt
EASI software was used to plot the calculated data.
Results. Distribution of different MWK species in
solution at equilibrium. We evaluated MLCK species
distribution by size using LCS method [18]. Fig. 1
shows typical pattern of such distribution for the
kinase solution at 4.9 /Ш concentration. More correct
data obtained as average of 17 measurements are
following:
where Z) a v
m, Dj, £>av° are average hydrodynamic
diameters of MLCK monomer, dimer and oligomer,
respectively.
Light scattering in the interval of 1266—5000 nm
370
DETECTION OF PROTEIN SPECIES BY LIGHT SCATTERING
Fig. 1. Particle size distribution of MLCK obtained by LCS method.
Experimental conditions: MLCK concentration 4.9 juM; buffer AA +
100 mM NaCl. Hydrodynamic diameter of particles Dzv (in nm) is
plotted in logarithmic scale for convenience. The Fig. shows data
obtained for one experiment. Averages for all data are given in text
occurs due to the presence of small quantity of the
dust particles, which are very difficult to remove
completely by the filtration. The other components
were related to the kinase particles, which was
confirmed by the dependence of their scattering on
the angle (data not shown). It should be pointed that
the regularisation program used in the LCS method
provides size for spherical particles. Therefore, hyd
rodynamic sizes obtained by this method for globular
proteins are close to their true sizes, that we were
able to confirm in control experiments with bovine
serum albumin. However, the hydrodynamic size
obtained for proteins differing strongly from sphere is
a rather conditional value. It applies in full measure
to MLCK which was evaluated by ultracentrifugation
method to be a rod-shaped molecule of 50 nm in
length and 2.2 nm in diameter [19]. Close by
dimentions (60 * 2 nm) is myosin subfragment-2
(SF2) [20]. SF2 hydrodynamic diameter was found
by the LCS method to be of 10.8 nm [21 ]. Accor
dingly, we identified particles with D a v = 7.5 nm in
6—9 nm interval (Fig. 1) as MLCK monomers,
possibly with some impurity of smaller species corres
ponding to the kinase larger proteolytic fragments.
The kinase dimer length is twice as that of the
monomer [19]. This was confirmed by the presence
of particles with D a v = 21.2 nm in the interval of
16—26 nm. The particles with £>av= 162.0 nm in
79—317 nm interval seem to represent the kinase
oligomer species. More correct D a v values, used in
further calculations, were obtained as an averages of
all experimental data (see above). They are 8.6, 20.2
and 158.3 for monomer, dimer and oligomer, res
pectively.
It must be emphasized that owing to the small
size of CaM molecules and their low concentration the
contribution of the light scattering from CaM was
negligible under our experimental conditions. The
results obtained showed that Ca 2 +-CaM binding to the
kinase at both high and low MLCK to CaM ratios had
practically no influence on the size of enzyme species
and their relative concentrations, which were almost
the same as for inactivated enzyme (apoenzyme).
Elution of different MLCK species from chroma
tographic columns. To get information about the
different kinase species we investigated light scat
tering of the kinase eluting from a short gel filtration
column using the FPLC set-up connected to the Wyatt
multi-angle light scattering photometer. The column
did not serve to separate the species but to produce
continuous distribution of the kinase at wide range of
its concentrations, besides removing the troublesome
dust particles. Fig. 2, A, shows characteristic elution
profiles obtained with the FPLC UV-monitor (curve
1) and Wyatt DAWN light scattering photometer
(curve 2). In the case of a monodisperse sample these
curves must coincide within some constant factor
[12]. In our case they have complex shape as a result
of several elution peaks overlapping. Using similar
plots obtained for monodisperse systems (bovine se
rum albumin, insulin, highly aggregated kinase pre
parations, polysterene of M r - 30 kDa and Mr =
= 200 kDa) we decomposed these plots into contri
butions from individual peaks (Fig. 2, B). Analogous
approach was used to decompose complex heat ab
sorption profiles into single components [22]. The
main requirements to be satisfied by this procedure
are the following: a) the sum of the monopeaks must
produce the original elution profile; b) the corres
ponding light scattering and UV-absorption mono-
peaks must coincide within some constant factor. As
shown in Fig. 2, B, UV-adsorption curve (J) is
decomposed into three overlapping monopeaks with
maxima at 3.8 ml (a 0), 5.0 ml (ad) and 6.4 ml (am).
We assigned these peaks, respectively, to the oligo-
meric, dimeric and monomelic kinase species. They
correspond to the overlapping monopeaks s°, sd and sm
of the light scattering profile. The peak sl clearly
results from the light scattering of the column «deb-
ris». It also appears under analogous conditions on
the elution profiles of bovine serum albumin, myosin,
myosin subfragment SI as well as other proteins.
There are small contributions of the elution plots for
which the monopeaks do not overlap and which,
therefore, correspond to the individual kinase species:
371
FILENKO A. M.
7Ш? /
Contribution of MLCK species to elution profiles for gel filtration
column
•Areas of individual monopeaks (see Fig. 2, B) were found by
weighting. Index «i» corresponds to о — oligomer, d — dimer or
m — monomer; **This value was estimated from UV absorption
monopeaks (a') taking into account that total area of all monopeaks
is 100 %; ***This parameter was calculated from the expression: 6l -
- (area sVarea a*)/(area sd/ area <zd); it represents light scattering
of MLCK oligomer and monomer relative to dimer assuming that
wt. % concentrations of all three species are equal.
Volume, ml
Fig. 2. Elution profiles obtained during MLCK gel filtration: A —
original curves (7, UV-absorption; 2 , light scattering); В— de
composition of elution profiles into monopeaks of absorption (a1) and
light scattering (sl) where index «o» represents the oligomer, «d» the
dimer and «m» the monomer; sl corresponds to light scattering of the
column «debris». Total mass of MLCK applied to the column was
0.56 mg (500 / i l ) . Flow rate was 0.25 ml/min
3.6—3.7 ml (oligomer), 5.1—5.5 ml (dimer) and
7.0—7.5 ml (monomer). These intervals can be used
to calculate the relative contributions of the individual
kinase species into the light scattering profiles.
More accurate results we obtained using in our
calculations corresponding monopeak area ratios
(Table 1). The table shows that, at the same weight
concentrations, the oligomer light scattering was 35
times larger than that of the dimer while the latter
about 3 times larger than that of the monomer. The
weight contributions of the kinase oligomer, dimer
and monomer in the eluate at the exit from the
column were 5.3, 81.5 and 13.2 % accordingly. Large
contribution of the oligomer into the light scattering
plot (Fig. 2, By curve 2) at relatively small weight
fraction is the result of its very high light scattering
level. These values together with the LCS data
obtained for the individual kinase species at equi
librium and the relative contributions dl of these
species into light scattering (see up) allows to calcu
late the relative content of the oligomers, dimers and
monomers at equilibrium (Table 2).
Mr and RMS radius for different MLCK species.
Fig. 3 shows the curves of molecular weight and root
mean square (RMS) radius vs. elution coordinate
which were calculated using Wyatt ASTRA and Wyatt
EASI software. These curves have characteristic 5-like
form. The data calculated in the elution interval of 5.5
to 7.0 ml are not reliable enough, especially for RMS
radius, owing to the poor light scattering by the
monomer particles. Due to the oligomer and dimer
peaks overlapping in the 3 to 5 ml interval the
calculations for each elution slice give weight-average
molecular weights and weight-average RMS radii.
These values change smoothly along the elution
profile accordingly to the weight ratio change of the
MLCK species.The characteristic feature of the curves
given in Fig. 3 is a rapid growth of the molecular
weight with a decrease in elution coordinate (by a
factor of 102 in the interval of 5 to 3.6 ml, Fig. 3, A)
at the relatively small RMS radius increase (22 to
80 nm, Fig. З, B) corresponding to such dramatic
change of molecular weight.
Using the intervals of elution profile corres
ponding to only single kinase species (Fig. 2, B) and
calculation data presented in Fig. 3 we determined the
molecular weights and RMS radii for the oligomer and
dimer MLCK species (Table 3). For the monomer we
372
DETECTION OF PROTEIN SPECIES BY LIGHT SCATTERING
Table 2
Percent content of different MLCK species in solution at
equilibrium
l.Oc+9
Kinase species Oligomer Dimer Monomer
•Data taken from text. For values of the index «/» values see Table
1; **Data taken from Table 1; ***Concentrations of different kinase
species at equilibrium in wt. % were calculated from the following
expression: В* - [(Іі/ді)'ЛҐ/д°+Ґі/дй+Іт/дт)] -100.
l.Oe+4 - T -
5.0 6.0
Volume, ml
Fig. 4. Elution profiles and Mr vs elution volume for different MLCK
to CaM molar ratio; 10:1 ( x ~ - x ) , 5:1 ( + - - + ) , 1:1.5 ( o - - o )
and without CaM (V — V) . Total injected mass of kinase in all
cases was 0.56 mg and 0,1 mM CaCl 2 was added to the elution
buffer
о ' ,
3.0 4.0 5.0 6.0 7.0
Volume, ml
Fig. 3. MLCK molecular mass (A) and RMS radius (B) vs elution
volume for gel-filtration column. Details see in the legend to Fig. 2
were able to calculate only Mr because of its poor light
scattering.
All the data given in Fig. 2 and 3 are obtained
for the inactive kinase. However it is worth noting
that kinase activation by binding to CaM at different
apoenzyme to CaM ratios doesn't influence practically
the elution profiles and the curves of the molecular
weight and RMS radius vs. elution coordinate (Fig.
4). This indicates that wt % ratio of the three kinase
species remains nearly unchanged during the activa
tion, which is in the agreement with the LCS data.
The same was also confirmed in the experiments
where CaM and kinase were syringed directly into the
photometer flow cell immediately after their mixing in
the presence of Ca 2 + (data not shown).
A number of experiments were carried out with a
CM-affinity column. In the presence of Ca 2 + ions
MLCK binds to CaM covalently linked to the column
matrix, and can be specifically eluted with a solution
containing EGTA. Fig. 5 shows the elution profiles
together with MT and RMS radius dependencies
calculated. As clear from Fig. 5, A, both elution
profiles (UV-absorption, curve / , and light scattering,
curve 2) nearly coincide indicating that the eluate
practically contains a single kinase species. Fig. 5, В
suggests that this species corresponds to the oligomer
with RMS radius of about 80 nm similar to the one
eluted from the gel-filtration column. In contrast, M f
373
FILENKO A. M.
Table 3
1.0 10'
4.0
Volume, ml
Fig. 5. MLCK M r (A) and RMS radius <#) vs elution coordinate for
CaM-affinity column. For convenience UV absorption (curve 1) and
light scattering (curve 2) profiles are also included. 0.53 mg (plus
0.1 mM CaCl 2 ) was applied to the column; elution solution contained
2 mM EGTA. Flow rate was 0.15 ml/min
of this oligomer was about 10 times less than M r of
that from the gel-filtration column.
Experiments with pause in the elution process. As
indicated in Materials and Methods the volume of
tubing connecting the gel-filtration column with the
flow cell of Wyatt photometer (delay volume) was
about 0.26 ml. This means that at the elution rate of
0.25 ml/min the portion eluating from the column
must reach the cell in about 1 min. As could be seen
from the above results the different MLCK species
separated by the column remained nearly unchanged
during this time. This means that mutual transition
between these species at the approach to the equilib
rium state is rather slow.
To have more detailed notion about the MLCK
species stability we carried out a number of expe
riments with the elution interruption. Fig. 6, A and B,
shows the results of two such experiments with the
pauses in the elution from the gel-filtration column at
4 and 5 ml. As can be seen in Fig. 2, B, at the elution
coordinate 5 ml practically pure dimer is eiuting from
the column.
Therefore the elution pause in this point let us
observe the process of dimer transition to the equi
librium state with the formation of two other kinase
species — monomer and oligomer. As Fig. 6, A, shows
this process continues for about 10 min indicating the
stability of the dimer. The oligomer seems to be still
more stable. Indeed, the elution pause at 4 ml, where
eluate contains equal weight amounts of dimers and
oligomer but light scattering is practically caused by
oligomer species (Fig. 2, B), gives the process of very
slow decrease of light scattering intensity which
indicates on the very slow transition of the oligomer
to the other kinase species (Fig. 6, B). Even after
20 min from the elution interaption the light scat
tering intensity is reduced only by about 11 % which
indicates the transition of only small amounts of
oligomer into other kinase species during this time.
Discussion. The light scattering data obtained in
this investigation show that in solution MLCK exists
as the equilibrium mixture of the oligomeric, dimeric
and monomelic species. The preliminary experiments
(not given here) revealed that the polymer species
percentage decreased at ionic strength increasing. At
the ionic strength of 60 mM (original buffer AA) the
kinase solution opalescences, which indicates a high
amount of the oligomer species. Upon the addition of
300 mM NaCl to the buffer almost only dimer and
monomer were present in the solution at the excess of
the latter. The ionic strength of 160 mM chosen in
our experiments turned out to be optimal for the
distinct separation of the individual MLCK species,
their percentage estimation in both equilibrium state
and the eluate and for their properties investigation.
According to our data (Table 3) the dimer
molecular mass is about 2.0-10 5 Da which is in good
agreement with the monomer molecular mass Mr =
374
DETECTION OF PROTEIN SPECIES BY LIGHT SCATTERING
Fig. 6. Time-dependent changes of MLCK light scattering after
interruption of the elution from gel-filtration column at 5 ml (A)
where practically pure dimer is present and at 4 ml (B) (oligomer +
dimer). For more details see text
- 1.06-105 Da calculated by Olson et al. [23] from
the amino acid sequence. Ausio et al. [19 ] have shown
by analytical ultracentrifugation that MLCK molecule
has the rod-like shape of length L = 50 nm, diameter
<i = 2.15 nm, the axial ratio 19 and RMS radius 7?g =
= 14.4 nm. By definition [12] RMS radius is
* , = V[ (1 /M) / rzdm], (1)
where the integration is over mass elements of the
particle with respect to the center of its gravity.
From (1) for rod-like particle we have
R = L/2V3", (2)
where L is the rod length. The axial ratio for MLCK
dimer is 32 [19 ]. This indicates that at the axial ratio
19 for the kinase monomer its dimer length is 1.2
times less than double length of the monomer. Hence
we may calculate RMS radius for the dimer Rg =
- (2Lm/1.2)/2 = 24 nm. In our experiments we ob
tained the close value of i? g = 22 nm (Table 3). The
above-mentioned authors have not discovered the
oligomer species since their investigations were made
at rather high ionic strength (0.2 M NaCl) and high
sucrose concentration. These factors strongly reduce
the ionic and hydrophobic interactions, which are
mainly responsible for the supramolecular structure
formation.
Taking into consideration relatively small in
crease of the oligomer RMS radius with respect to that
of the dimer (about 4-fold) at about 102-fold dif
ference in their molecular masses (Fig. 3) we may
suggest several models for the oligomer structure
organization.
Ring structure^ where MLCK molecules are as
sociated in head-to-tail fashion (Fig. 7, A). It is not
difficult to show using equation (1) that for the ring,
thickness of which may be neglected, RMS radius is
determined by the expression
Rg = V ( * 2 +AV12) , (3)
where R and h are the ring radius and height
respectively. Hence even at h = R/2 we have # g =
- 1.01Л, so in our reasoning we may suppose with
sufficient accuracy that RMS radius of the MLCK ring
structure (Rg = 80 nm) is equal to its geometrical
radius. Therefore at the association of the kinase
molecules without overlapping the ring must contain
(biRg/Lm) = 2л -80/50 « 10 molecules. At the mono
mer molecular mass of 1.06 105 kDa the molecular
mass of such ring structure must be Nf. ~ 106 Da. We
obtained for MLCK oligomer Mr° « 10 у Da (Table 3).
This suggests that the ring structure must be turned
up into a helix with a number of turns n = 10. At such
number of turns and the MLCK monomer diameter
equal 2.15 nm the helix height at the compact packing
is supposed to be /1 = 2.15-10 = 22 nm. It must be
noted that such helix structure is much closer to a
spherical form than the rod-like monomer and dimer.
It might be the reason why the oligomer size obtained
by the LCS method (158 nm, see up) corresponds
rather well to the size obtained by the Wyatt DAWN
photometer measurements (D = 2Rg° = 160 nm).
Rod-like structure may also account for the data
obtained (Fig. 7, B). Indeed at i?g° = 80 nm we may
find from (2) the rod length L = 2V3Rg°~ 280 nm,
which at the monomer length 50 nm [19] correlates
well with 6 kinase molecules (hexamer) arranged in
line forming one «building unit». Molecular mass of
the oligomer at the exit from gel-filtration column is
Mr° = 107 Da, which corresponds to about 100 mono
mer molecules. Accordingly at the rod-like oligomer
structure the last must contain about 17 such building
units arranged in parallel. In the oligomer with Mr° =
375
HLENKO A. M.
A
258 nm
Fig. 7. Possible models of MLCK oligomer organization: ring
structure 04 ) , rod-like structure (/?), and spiral structure (C).
Details see in the text
= 2.4 106 Da at the exit from CaM affinity column
(Fig. 5, A) there should be only four such building
units with 6 molecules.
Spiral structure (Fig. 7, C). The 6-molecule unit
may be also interpreted as an extended spiral struc
ture. From (1) we can get for a spiral
Rg = V [ * 2 + (dV)Vl2T = V(i? 2 + # V 1 2 ) , (4)
where R, c, N and H are radius, pitch, number of
turns, and height of a spiral, respectively. Such
6-molecule spiral structure with R- 10 nm and # =
= 258 nm (Fig. 7, C) fits well to smooth myosin
filament with which the kinase is tightly associated.
We used this spiral structure model to interpret a
possible location of the kinase molecules on myosin
filaments in vivo (see details in [24]). In solution
such extended spiral structures should get together
into the associates analogous to the above described
rod-like model. In case of the rod-like or spiral
structures it is reasonable to suggest that 6 molecule
unit is a real oligomer and that the structures
registered by light scattering are the aggregates
formed by these unites.
Attention should be paid to the strong increase in
the supramolecular kinase species in eluate (Table 1)
in comparison with their content in the equilibrium
state (Table 2). This may be explained by the fact
that translational motility of eluting protein molecules
in rather small cells of the column bed is restricted.
Consequently the main entropy factor preventing the
weak interactions, which are basically responsible for
the formation of supramolecular structures [1 ], is
reduced essentially. Analogous role in restriction of
translational motility of protein molecules must be
fulfilled by cytoplasmic structures of living cell. The
refore this experimental fact may point indirectly to a
significant role of the oligomeric and dimeric species
in vivo.
It is of interest to compare the results obtained
at the elution pauses (Fig. 6) with the calculated data.
At the elution interuption in the point 5 ml practically
pure MLCK dimer is present in the eluate at the
column outlet (Fig. 2, A). Light scattering registration
of the eluate portion, which reaches DAWN photo
meter flow cell, begins in about 1 min from the
moment of its exit from the column. Exponential
extrapolation of the curve presented in Fig. 6, A, to
the moment of the eluate exit results in the original
light scattering level (corresponding to the pure
dimer) / 0 = 0.160. This curve goes up to the constant
light scattering level 7C = 0.215. So the gain of light
sca t t e r ing in t ens i ty must be Д / = [(0.215—
0.160)/0.160]100 = 34 %. This level corresponds to
the attainment of the equilibrium between three
kinase species. It is not difficult to see that i-Xh kinase
species gives contribution into light scattering
(0.160-2?-6 і)/100 (see Table 2). Hence individual
contributions for the oligomer, dimer and monomer
must be 0.117, 0.085 and 0.025, respectively, which
gives in sum the level of scattering at equilibrium
0.227. This corresponds to the total gain against the
original level [(0.227—0.160) /0.160 ] 100 = 42 %
which rather well agrees with the experimental value
of 34 %. The discrepancy in these values may be due
to some inaccuracy of the data obtained by the LCS
method, which were used for calculation of values B1,
not high accuracy of the determination of the mono-
376
DETECTION OF PROTEIN SPECIES BY LIGHT SCATTERING
mer contribution dm into light scattering and possibly
to some instability of DAWN photometer in the
conditions of flow interaption.
Fig. 6 shows that the MLCK oligomers and
dimers are long living stable structures. We suppose
that the oligomer assemblage goes through association
of individual MLCK molecules in head-to-tail fashion.
If the orientation of the MLCK molecules in the dimer
was the same being the first step on the pathway of
oligomer assembling it would be hard to understand
why other intermediate forms such as trimer, tetra-
mer etc. could not be discovered.
The most competent explanation of this ob
servation is that the dimers of head-to-tail fashion are
unstable, transitional, short-living species on the
pathway of oligomer assembling while the long-living
dimers discovered in solution are formed by the
MLCK molecules associated in tail-to-tail or head-to-
head fashion. Thus we can suppose that the most
probable scheme of the mutual species transition is:
Oligomer о Monomer Dimer and therefore the for
mation of the dimer and oligomer species must go
through the step of monomer.
Conclusions and remarks. The main results of
our investigations are the following:
1. In solution under conditions close to phy
siological the smooth muscle myosin light chain
kinase exists as a mixture of monomers, dimers and
oligomers. At equilibrium the contribution of these
forms in weight percents is 45, 53 and 2, respectively.
Such proportion of different kinase species is charac
teristic for both apoenzyme and activated kinase. An
important point is that supramolecular MLCK species
in the eluate were strongly increased in comparison
with their content in equilibrium state. The contri
butions of the kinase oligomer, dimer and monomer
in the eluate at the exit from gel-filtration column
were 5.3, 81.5 and 13.2 wt. % accordingly. At the exit
from CaM-affinity column we obtained practically
pure oligomer. Such oligomer in comparison with the
gel- filtration one had 3 times less Mr and its time of
transition to the equilibrium state was significantly
less.
2. All three kinase species are characterized by
prolonged lifetime. The transfer from the state of pure
dimer into equilibrium state lasts about 10 min. The
same is true for the oligomer at the exit from
CaM-affinity column. But the oligomer with much
greater M r (or more exactly oligomer aggregates) at
the exit from gel-filtration column is much more
stable and transfer from it to the equilibrium lasts for
more extended time.
3. Oligomer is characterized by RMS radius Rg
~ 80 nm. The oligomer structure may be presented
as a helical ring containing about 10 kinase molecules
per turn with a number of turns about 10 (data for
gel-filtration column). Another possible and more
realistic explanations of the data obtained involves the
rod-like or spiral models according to which 6 kinase
molecules arranged in line or prolonged spiral form a
structural unit which must be a real oligomer (hexa
mer). About 17 such structural units associated in
parallel may form aggregates with molecular mass of
about 107 kDa. The kinase elongated spiral hexamer
fits well to the structure of smooth myosin filament
with which the kinase is in close contact in vivo [25,
26].
The possibilities of the light scattering methods
used and peculiarities of their usage.
a) LCS allows to discover particles with different
molecular masses, find out their percent contribution
into intensity of light scattering and determine the
dimensions of spherical particles. For spherical parti
cle the average diameter Z)av obtained by this method
is equal to the diameter of hydrated molecule. In our
case of rod-like particles £>av presents some average
value which is far from real dimensions of these
molecular forms. The most, which we could get by
only this approach, was the quantity of particle
species in solution, contribution of different species
into light scattering and the fact that dimer dimension
is twice that of the monomer. We were able to find
out wt. % equilibrium distribution for different kinase
species only by comparison the LCS data with the
results of multi-angle light scattering analysis of the
kinase eluate.
b) Multi-angle light scattering photometry in
conjunction with FPLC permited to determine mo
lecular masses and RMS radii for the kinase dimer
and oligomer (for monomer only RMS radius due to
low light scattering), wt.% distributions of different
species in eluate, specific (per mass unit) light
scattering of different species (which together with
the LCS data allowed to estimate weight ratio for
different kinase species in equilibrium solution).
c) LCS and multi-angle light scattering used
together give more complete information about the
pattern of different particle distributions in solution
and characterization of these particles.
d) The procedure of decomposition of mass
sensitive and light scattering elution profiles into
individual monopeaks used in this study for the first
time enables more careful analysis of the data ob
tained.
e) Interruption of elution at specified points used
in this study allows to evaluate the time of transfer of
different protein species to the equilibrium state.
377
FILENKO A. M.
M. ФІЛЄНКО
Д о с л і д ж е н н я о л і г о м е р н и х ф о р м б ілків м е т о д а м и
світлорозсіювання: надмолекулярні структури кінази легких
ланцюгів міозину гладеньких м'язів
Резюме
Сучасні методи багатокутового світлорозсіювання в поєд
нанні зі швидкісною хроматографією білків та лазерною
кореляційною спектроскопією дають досить детальну інфор
мацію щодо розподілу білкових частинок у розчині, їхнього
розміру та молекулярної маси. Дані, отримані при дослід
женні кінази легких ланцюгів міозину гладеньких м'язів, свід
чать про те, що цей білок існує в розчині як рівноважна суміш
олігомерних, дймерних та мономерних часток у кількісному
співвідношенні 2, 53 та 45 вагових % відповідно. На виході з
гель-фільтраційної колонки рівновага значно зсунута в бік
олігомерних форм кінази і час переходу до рівноважного стану
становить приблизно 10 хв. Димер кінази має стрижнсвидну
структуру з середньоквадратичним радіусом (СКР) біля 22
нм. Для олігомеру СКР складає біля 80 нм. Його структуру
можна представити у вигляді спірального кільця із 10 витків
з 10 молекулами кінази на виток. Структуру олігомера добре
описують також стрижневидна або спіралевидна моделі з
шістьма молекулами, розміщеними вздовж лінії або витягну
тої спіралі. Біля 17 таких шестимолекулярних елементів
утворюють паралельно асоційовані агрегати. Попередні до
слідження показали, що низка інших білків також існує в
розчині як рівноважна суміш мономерів і надмолекулярних
структур з тривалим часом життя.
А. М. Филенко
Исследование олигомерных форм белков методами
светорассеяния: надмолекулярные структуры киназы легких
цепей миозина гладких мышц
Резюме
Современные методы многоуглового свсеторассеяния совме
стно со скоростной хроматографией белков и лазерной корре
ляционной спектрскопией дают достаточно полную информа
цию о распределении белковых частиц в растворе, их размере
и молекулярной массе. Данные, полученные при исследовании
киназы легких цепей миозина гладких мышц, свидетельству
ют, что этот белок существует в растворе как равновесная
смесь олигомерных, димерных и мономерных частиц в количе
ственном соотношении 2, 53 и 45 весовых % соответственно.
На выходе из гель-фильтрационной колонки равновесие сильно
сдвинуто в сторону олигомерных форм киназы и время пере
хода в равновесное состояние составляет приблизительно 10
мин. Димер киназы имеет стержневидную структуру со сред
неквадратичным радиусом (СКР) около 22 нм. Для олигомера
СКР составляет около 80 нм. Его структуру можно предста
вить в виде спирального кольца из 10 витков с 10 молекулами
киназы на виток. Структуру олигомера хорошо описывают
также стержневидная или спиралевидная модели с шестью
молекулами, размещенными вдоль линии или вытянутой спи
рали. Около 17 таких шестимолекулярных элементов образу
ют параллельно ассоциированные агрегаты. Предварительные
исследования показали, что ряд других белков также сущест
вует в растворе как равновесная смесь мономеров и надмоле
кулярных структур с продолжительным временем жизни.
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