Chromophore Protonation States and the Proton Shuttle Mechanism in Green Fluorescent Protein: Inferences Drawn from ab Initio Theoretical Studies of Structures and Infrared Absorption SpectraHi-Young Yoo, J.A. Boatz, Volkhard Helms, J. Andrew McCammon and Peter W. LanghoffJournal of Physical Chemistry B, Vol. 105, No. 14, pp. 2850-2857 (2001)
Assignments are provided of prominent features in the recently measured
Fourier transform infrared (FTIR) difference spectra of green fluorscent
and photoactive yellow proteins (GFP, PYP) employing ab inito
calculations of the ground electronic state structures and vibrational
spectra of their chromophores in selected protonation states. Particular
attention is addressed to inferring the protonation states of wild-type
GFP chromophore and to reconciling the measured FTIR difference spectrum
with a proposed proton shuttle mechanism in which protonated and
deprotonated forms of the chromophore are paired with corresponding
charge states of a Glu222 residue shuttle terminus. The calculated GFP
IR difference spectrum obtained from the neutral-anionic pair of
chromophores is found to be in general accord with the FTIR measurements
on wild-type GFP in its protonated and deprotonated forms, whereas the
spectrum obtained from the zwitterionic-cationic pair of chromophores
provides a less satisfactory simulation of the data. The apparent
absence of a carbonyl band in the measured GFP FTIR difference spectrum,
a feature expected upon protonation of the carboxylic Glu222 residue, is
reconciled by the presence of a carbonyl mode in the imidazole ring of
the neutral chromophore which partially obscures the anticipated R-COOH
Glu222 feature in the calculated spectrum. By contract, the
corresponding carbonyl mode in the PYP chromophore is predicted to be
significantly weaker and at lower frequency than in GFP, accounting in
part for the presence of an unobscured prominent R-COOH Glu46 residue
carbonyl mode in the measured PYP FTIR difference spectrum. Accordingly,
the present ab initio theoretical study supports the predominance of
neutral and anionic forms of wild-type GFP chromophore, and it argueably
reconciles the available FTIR data with a probable Glu222 terminus of
the proposed proton shuttle mechanism in the protein. Additional
experimental studies of IR and Raman difference spectra in GFP and PYP,
including particularly isotopic substitutions, are suggested to
complement additional theoretical studies in progress.
The Adaptive Multilevel Finite Element Solution of the Poisson-Boltzmann Equation on Massively Parallel ComputersN.A. Baker, D. Sept, M.J. Holst and J.A. McCammonIBM Journal of Research and Development, Vol. 45, No. 3-4, pp. 427-438 (2001)
Using new methods for the parallel solution of elliptic partial
differential equations, the teraflops computing power of massively
parallel computers can be leveraged to perform electrostatic
calculations on large biological systems. This paper describes the
adaptive multilevel finite element solution of the Poisson-Boltzmann
equation for a microtubule on the NPACI IBM Blue Horizon supercomputer.
The microtubule system is 40 nm in length, 24 nm in diameter, consists
of roughly 600000 atoms, and has a net charge of -1800 e.
Poisson-Boltzmann calculations are performed for several processor
configurations and the algorithm shows excellent parallel scaling.
Load Balancing of Molecular Dynamics Simulation with NWChemT.P. Straatsma and J.A. McCammonIBM Systems Journal, Vol. 40, No. 2, pp. 328-341 (2001)
NWChem is a computational chemistry software suite developed for
massively parallel computers in the W. R. Wiley Environmental Molecular
Sciences Laboratory at the U.S. Department of Energy's Pacific Northwest
National Laboratory software computational chemistry applications
classical molecular dynamics quantum mechanical calculations
architecture memory domain decomposition intermolecular interactions
periodic atomic reassignmentsoratory. This software integrates a range
of modules for computational chemistry applications, including classical
molecular dynamics simulations and quantum mechanical calculations. This
contribution provides details of the classical molecular dynamics module
and focuses on issues related to load balancing on massively parallel
computers, in particular the IBM SP and the Cray T3E as examples of
distributed and shared memory massively parallel architectures. The
implementation of the molecular dynamics module of NWChem is based on a
domain decomposition of the chemical system, taking advantage of the
distribution of data to reduce the memory requirements and the locality
of intermolecular interactions to reduce the communication requirements.
This approach results in a more complex implementation because of the
requirement of periodic atomic reassignments and the need for
sophisticated load-balancing techniques.
A Model for Enzyme-Substrate Interactions in Alanine RacemaseMary Jo Ondrechen, James M. Briggs and J. Andrew McCammonJournal of the American Chemical Society, Vol. 123, No. 12, pp. 2830-2834 (2001) [PubMed 11456969]We report on a theoretical model for the complex of the enzyme alanine
racemase with its natural substrate (L-alanine) and cofactor (pyridoxal
5'-phosphate). Electrostatic potentials were calculated and ionization
states were predicted for all the ionizable groups in alanine racemase.
Some rather unusual charge states were predicted for certain residues.
Tyr265' has an unusually low predicted pK
a of 7.9 and at pH
7.0 has a predicted average charge of -0.37, meaning that 37% of the
Tyr265' residues in an ensemble of enzyme molecules are in the phenolate
form. At pH 8-9, the majority Tyr265' side groups will be in the
phenolate form. This lends support to the experimental evidence that
Tyr265' is the catalytic base involved in the conversion of L-alanine to
D-alanine. Residues Lys39 and Lys129 have predicted average charges of
+0.91 and +0.14 respectively at pH 7.0. Lys39 is believed to be the
catalytic base for the conversion of D-alanine to L-alanine and the
present results show that, at least some of the time, it is in the
unprotonated amine form and thus able to act as a base. Cys311', which
is located very close to the active site, has an unusally low predicted
pK
a of 5.8 and at pH 7.0 has a predicted average charge of
-0.72. It appears that the enzyme has the ability to stabilize negative
charge in the region of the active site. Implications for selective
inhibitor design are discussed.
Calculation of Weak Protein-Protein Interactions: The pH Dependence of the Second Virial CoefficientAdrian H. Elcock and J. Andrew McCammonBiophysical Journal, Vol. 80, No. 2, pp. 613-625 (2001) [PubMed 11159430]Interactions between proteins are often sufficiently weak that their
study through the use of conventional structural techniques becomes
highly problematic. Of the few techniques capable of providing
experimental measures of weak protein-protein interactions perhaps the
most useful is the second virial coefficient B
22, which
quantifies a protein solution's deviations from ideal behaviour. It has
long been known that B
22 can in principle be computed, but
only very recently has it been demonstrated that such calculations can
be performed using protein models of true atomic detail
(http://dx.doi.org/10.1016/S0006-3495(98)77691-X" target="_blank"
class="ref">Neal et al., Biophys. J. 1998, 75:2469-2477). The work
reported here extends these previous efforts in an attempt to develop a
transferable energetic model that is capable of reproducing the
experimental trends obtained for two different proteins over a range of
pH and ionic strength. We describe protein-protein interaction energies
by a combination of three separate terms: (i) an electrostatic
interaction term based on the use of effective charges, (ii) a term
describing the electrostatic desolvation that occurs when charged groups
are buried by an approaching protein partner, and (iii) a solvent
accessible surface area (SASA) term that is used to describe
contributions from van der Waals and hydrophobic interactions. The
magnitude of the third term is governed by an adjustable parameter,
gamma, that is altered to optimize agreement between calculated and
experimental values of B
22. The model is applied separately
to the proteins lysozyme and chymotrypsinogen, yielding optimal values
of gamma that are almost idential. However, there are clear difficulties
in reproducing B
22 values at extremes of pH. Rigorous
calculations of the protonation states of charged groups in the 200 most
favourable structures suggest that these difficulties are due to a
neglect of the ionization state changes that often accompany
complexation. Despite this problem, the fact that identical gamma values
are obtained from two different proteins suggests that the energetic
model developed here may well be transferable to other protein systems.
Since the model proposed is extremely rapid to evaluate, it can be used
in dynamical simulations of weak protein-protein interactions.
Statistical Analysis of the Fractal Gating Motions of the Enzyme AcetylcholinesteraseT.Y. Shen, Kaihsu Tai and J. Andrew McCammonPhysical Review E, Vol. 63, article 041902, 6 pages (2001) [PubMed 11308872]The enzyme acetylcholinesterase has an active site that is accessible
only by a "gorge" or main channel from surface, and perhaps by secondary
channels such as the "back door". Molecular dynamics simulations show
that these channels are too narrow most of the time to admit substrate
or other small molecules. Binding of substrate is therefore "gated" by
structural fluctuations of the enzyme. Here, we analyze the fluctuations
of these possible channels, as observed in the 10.8 ns trajectory of the
simulation. The probability density function of the gorge proper radius
(defined in text) was calculated. A double-peak feature of the function
was discovered and therefore two states with a threshold were
identified. The relaxation (transition probability) functions of these
two states were also calculated. The results revealed a power-law decay
trend and an oscillation around it, which show properties of fractal
dynamics with a "complex exponent". The cross-correlation of potential
energy versus proper radius was also investigated. We discuss possible
physical models behind the fractal protein dynamics; the dynamic
hierarchical model for glassy systems is evaluated in detail.
Historical Overview and Future ChallengesJ. Andrew McCammonIn "Free Energy Calculations in Rational Drug Design," M.R. Reddy and M.D. Erion, Eds., Kluwer Publishing, The Netherlands, pp. 1-6 (2001)
The selective binding of molecules to form productive complexes is of
central importance to pharmacology and medicinal chemistry. Although
kinetic factors can influence the yields of different molecular
complexes in cellular and other non-equilibrium environments, the
primary factors that one must consider in the analysis of molecular
recognition are thermodynamic. In particular, the equilibrium constant
for the binding of molecules A and B to form the complex AB depends
exponentially on the standard free energy change associated with
complexation. Here, I provide a brief review of the history and new
directions of free energy calculations.
The X-ray Crystal Structure of an SR Protein Kinase in Yeast (Sky1p) Reveals a Novel Mechanism for Constitutive ActivityBrad Nolen, Chi Y. Yun, Chung F. Wong, J. Andrew McCammon, Xiang-Dong Fu and Gourisankar GhoshNature Structural Biology, Vol. 8, pp. 176-183 (2001) [PubMed 11175909]The SR proteins and RS domain-containing proteins are a class of
splicing factors rich in arginine-serine (RS) dipeptides that are
phosphorylated by the SR protein kinases (SRPHs). SRPKs are
constitutively active and display remarkable substrate specificity.
Recently, the sole
Saccharomyces cerevisiae SRPH family member,
Sky1p, was shown to regulate nuclear import of the shuttling RNA binding
protein Npl3p, which has been implicated in the mRNA export. Here we
present the three-dimensional structure of a fully active truncated
Sky1p. Analysis of the structure and structure-based functional studies
reveal that the carboxyl-terminal tail, an unusual glutamine residue
located in the P+1 loop, and a unique mechanism for the positioning of
helix αC act together to render Sky1p constitutively active. We
have modeled a Npl3p-derived substrate peptide bound to Sky1p. The model
complex combined with mutagenesis studies illustrate the molecular basis
for substrate recognition by this kinase, and suggest a mechanism for
SRPKs to catalyze a novel sequential phosphorylation reaction on the RS
dipeptide repeats characteristic of mammalian SR proteins.
Computer Simulation of Protein-Protein InteractionsAdrian H. Elcock, David Sept and J. Andrew McCammonJournal of Physical Chemistry B, Vol. 105, No. 8, pp. 1504-1518 (2001)
The use of computer simulations in investigations of protein-protein
interactions is discussed. First, crystallographic analyses of known
protein-protein complexes are summarized with particular emphasis being
placed on the atomic nature of the interactions. Models available for
describing macromolecular association energetics are then discussed,
with special reference to the treatment of electrostatic and nonpolar
interactions. The use of these models in combination with efficient
search methods is discussed in context of the so-called protein docking
problem and in the description of weaker (i.e., noncrystallisable)
protein-protein interactions. Finally, simulations of the dynamics of
protein-protein association events are outlined. In all cases,
differences are stressed between the atomically detailed view of
protein-protein interactions and the view implicit in the use of simpler
colloidal models.
Identification of Protein Oligomerisation States by Analysis of Interface ConservationAdrian H. Elcock and J. Andrew McCammonProceedings of the National Academy of Sciences of the USA, Vol. 98, No. 6, pp. 2990-2994 (2001) [PubMed 11248019]The discrimination of true oligomeric protein-protein contacts from
non-specific crystal contacts remains problematic. Criteria that have
been used previously base the assignment of oligomeric state on
consideration of the size of the interface area and/or the results of a
scoring function based on statistical potentials. Both techniques have a
high success rate, but fail in more than 10% of cases. More importantly,
the oligomeric states of several proteins are incorrectly assigned by
both methods. Here we test the hypothesis that true oligomeric contacts
should be identifiable based on an increased degree of conservation of
the residues involved in the interface. By quantifying the degree of
conservation of the interface and comparing it with that of the
remainder of the protein surface, we develop a new criterion that
provides a highly effective complement to existing methods.
Computational Analysis of PKA-Balanol InteractionsChung F. Wong, Philippe H. Hünenberger, Pearl Akamine, Narendra Narayana, Tom Diller, J. Andrew McCammon, Susan Taylor and Nguyen-Huu XuongJournal of Medicinal Chemistry, Vol. 44, No. 10, pp. 1530-1539 (2001) [PubMed 11334563]Protein kinases are important targets for designing therapeutic drugs.
This paper illustrates a computational approach to extend the usefulness
of a single protein-inhibitor structure in aiding the design of protein
kinase inhibitors. Using the complex structure of the catalytic subunit
of PKA (cPKA) and balanol as a guide, we have analyzed and compared the
distribution of amino acid types near the protein-ligand interface for
nearly 400 kinases. This analysis has identified a number of sites that
are more variable in amino acid types among the kinases analyzed, and
these are useful sites to consider in designing specific protein kinase
inhibitors. On the other hand, we have found kinases whose
protein-ligand interfaces are similar to that of the cPKA-balanol
complex and balanol can be a useful lead compound for developing
effective inhibitors for these kinases. Generally, this approach can
help us discover new drug targets for an existing class of compounds
that have already been well characterized pharmacologically. The
relative significance of the charge/polarity of residues at the
protein-ligand interface has been quantified by carrying out
computational sensitivity analysis in which the charge/polarity of an
atom or functional group was turned off/on, and the resulting effects on
binding affinity have been examined. The binding affinity was estimated
by using an implicit-solvent model in which the electrostatic
contributions were obtained by solving the Poisson equation and the
hydrophobic effects were accounted for by using surface-area dependent
terms. The same sensitivity analysis approach was applied to the ligand
balanol to develop a pharmacophoric model for searching new drug leads
from small-molecule libraries. To help evaluate the binding affinity of
designed inhibitors before they are made, we have developed a
semiempirical approach to improve the predictive reliability of the
implicit-solvent binding model.
Analysis of a Ten-nanosecond Molecular Dynamics Simulation of Mouse AcetylcholinesteraseKaihsu Tai, Tongye Shen, Ulf Börjesson, Marios Philippopoulos and J. Andrew McCammonBiophysical Journal, Vol. 81, No. 2, pp. 715-724 (2001) [PubMed 11463620]A 10 ns molecular dynamics simulation of mouse acetylcholinesterase was
analysed, with special attention paid to the fluctuation in the width of
the gorge, and opening events of the back door. The trajectory was first
verified to ensure its stability. We defined the gorge proper radius as
the measure for the extent of the gorge opening. We developed an
expression of an inter-atom distance representative of the gorge proper
radius in terms of projections on the principal components. This
revealed the fact that collective motions of many scales contribute to
the opening behavior of the gorge. Covariance and correlation results
identified the motions of the protein backbone as the gorge opens. In
the back door region, sidechain dihedral angles that define the opening
were identified. Additional data from this molecular dynamics simulation
can be found at http://mccammon.ucsd.edu/" target="_blank"
class="ref">http://mccammon.ucsd.edu/.
Thermodynamics and Kinetics of Actin Filament NucleationDavid Sept and J. Andrew McCammonBiophysical Journal, Vol. 81, No. 2, pp. 667-674 (2001) [PubMed 11463615]We have performed computer simulations and free energy calculations to
determine the thermodynamics and kinetics of actin nucleation and thus
identify a probable nucleation pathway and critical nucleus size. The
binding free energies of structures along the nucleation pathway are
found through a combination of electrostatic calculations and estimates
of the entropic and surface area contributions. The association kinetics
for the formation of each structure are determined through a series of
Brownian dynamics simulations. The combination of the binding free
energies and the association rate constants determines the dissociation
rate constants, allowing for a complete characterization of the
nucleation and polymerization kinetics. The results indicate that the
trimer is the size of the critical nucleus, and the rate constants
produce polymerization plots that agree very well with experimental
results over a range of actin monomer concentrations.
Electrostatics of Nanosystems: Application to Microtubules and the RibosomeNathan A. Baker, David Sept, Simpson Joseph, Michael J. Holst and J. Andrew McCammonProceedings of the National Academy of Sciences of the USA, Vol. 98, No. 18, pp. 10037-10041 (2001) [PubMed 11517324]Evaluation of the electrostatic properties of biomolecules has become a
standard practice in molecular biophysics. Foremost among the models
used to elucidate the electrostatic potential is the Poisson-Boltzmann
equation; however, existing methods for solving this equation have
limited the scope of accurate electrostatic calculations to relatively
small biomolecular systems. Here we present the application of numerical
methods to enable the trivially parallel solution of the
Poisson-Boltzmann equation for supramolecular structures that are orders
of magnitude larger in size. As a demonstration of this methodology,
electrostatic potentials have been calculated for large microtubule and
ribosome structures. The results point to the likely role of
electrostatics in the variety of activities of these structures.
Ordered Water and Ligand Mobility in the HIV-1 Integrase-5CITEP Complex: A Molecular Dynamics StudyHaihong Ni, Christoph A. Sotriffer and J. Andrew McCammonJournal of Medicinal Chemistry, Vol. 44, No. 19, pp. 3043-3047 (2001) [PubMed 11543671]A 2 ns molecular dynamics simulation has been carried out for the HIV-1
integrase-5CITEP complex in order to understand the role of water in
defining the ligand's binding mode and to address issues of binding site
flexibility and ligand motion. Although the ligand retains considerable
mobility within the active site, a structural water molecule bridging
5CITEP with Asp 64 and Asn 155 is identified in the simulation.
Consideration of this water molecule could open a route to new HIV-1
integrase inhibitors.
Atomistic Brownian Dynamics Simulation of Peptide PhosphorylationTongye Shen, Chung F. Wong and J. Andrew McCammonJournal of the American Chemical Society, Vol. 123, No. 37, pp. 9107-9111 (2001) [PubMed 11552818]We report the implementation of an all-atom Brownian dynamics simulation
model of peptides using the constraint algorithm LINCS. The algorithm
has been added as a part of UHBD. It uses adaptive time steps to achieve
a balance between computational speed and stability. The algorithm was
applied to study the effect of phosphorylation on the conformational
preference of the peptide Gly-Ser-Ser-Ser. We find that the middle
serine residue experiences considerable conformational change from
C
7eq to the α
R structure upon
phosphorylation. NMR
3J coupling constants were also computed
from the Brownian trajectories using the Karplus equation. The
calculated
3J results agree reasonably well with experimental
data for the phosphorylated peptide but less so for doubly charged
phosphorylated one.
Native State Conformational Dynamics of GART: A Regulatory pH-Dependent Coil-Helix Transition Examined by Electrostatic CalculationsDimitrios Morikis, Adrian H. Elcock, Patricia A. Jennings and J. Andrew McCammonProtein Science, Vol. 10, Issue 11, pp. 2363-2378 (2001) [PubMed 11604542]Glycinamide ribonucleotide transformylase (GART) undergoes a
pH-dependent coil-helix transition with pK
a = 7. An
α-helix is formed at high pH spanning eight residues of a
twenty-one residue long loop, comprising the segment
Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand
the electrostatic nature of this loop-helix, pK
a values of
all ionizable residues of GART have been calculated,using
Poisson-Boltzmann electrostatic calculations and crystallogrphic data.
Crystallographic structures of high and low pH E70A GART have been used
in our analysis. Low pK
a values of 5.3, 5.3, 3.9, 1.7, and
4.7, have been calculated for five functionally important histidines,
His108, His119, His121, His132, and His137, respectively, using the high
pH E70A GART structure. Ten theoretical single and double mutants of the
high pH E70A structure have been constructed to identify pair-wise
interactions of ionizable residues, which have aided in elucidating the
multiplicity of electrostatic interactions of the activation loop-helix,
and the impact of the activation helix on the catalytic site. Based on
our pK
a calculations and structural data, we propose that:
(1) His121 forms a molecular switch for the coil-helix transition of the
activation helix, depending on its protonation state, (2) a strong
electrostatic interaction between His132 and His121 is observed, which
can be of stabilizing or destabilizing nature for the activation helix,
depending on the relative orientation and protonation states of the
rings of His121 and His132, (3) electrostatic interactions involving
His119 and Arg122 play a role in the stability of the activation helix,
and (4) the activation helix contains the helix promoting sequence
Arg122-Gln123-Ala125-Leu125-Glu126, but its alignment relative to the
amino and carboxy termini of the helix is not optimal, and possibly of
destablizing nature. Finally, we provide electrostatic evidence that the
formation and closure of the activation helix creates a hydrophobic
environment for catalytic site residue His108, to facilitate catalysis.
Proton Transfer Dynamics of GART: The pH-Dependent Catalytic Mechanism Examined by Electrostatic CalculationsDimitrios Morikis, Adrian H. Elcock, Patricia A. Jennings and J. Andrew McCammonProtein Science, Vol. 10, Issue 11, pp. 2379-2392 (2001) [PubMed 11604543]The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes
the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to
glycinamide ribonucleotide (GAR), a process that is pH-dependent with
pK
a of = 8. Experimental studies of pH-rate profiles of wild
type and site directed mutants of GART, have led to the proposal that
HIS108, Asp144 and GAR are involved in catalysis, with His108 being an
acid catalyst, while forming a salt bridge with Asp144, and GAR being a
nucleophile to attack the formyl group of fTHF. This model implied a
protonated histidine with pK
a of 9.7 with a neutral GAR with
pK
a of 6.8 (http://dx.doi.org/10.1021/bi9904609"
target="_blank" class="ref">Shim J.H. and Benkovic, S.J., 1999,
Biochemistry, 38, 10024-10031). These proposed unusual
pK
a's have led us to investigate the electrostatic
environment of the active site of GART. We have used Poisson-Boltzmann
based electrostatic methods to calculate the pK
a's of all
ionizable groups, using the crystallographic structure of a ternary
complex of GART involving the pseudo-substrate 5-deaza-5, 6, 7, 8-THF
(5dTHF) and substrate GAR. Theoretical mutation and deletion analogs
have been constructed to elucidate pair-wise electrostatic interations
between key ionizable sites within the catalytic site. Also, a construct
of a more realistic catalytic site including a re-constructed
pseudo-cofactor with an attached formyl group, in an environment with
optimal local van der Waals interactions (locally minimized) that
imitates closely the catalytic reactants, has been used for
pK
a calculations. Strong electrostatic coupling among
catalytic residues His108, Asp144, and substrate GAR was observed, which
is extremely sensitive to the initial protonation and imidazole ring
flip state of His108 and small structural changes. We demonstrate that a
proton can be exchanged between GAR and His108 depending on their
relative geometry and their distance to Asp144, and when the proton is
attached on His108 catalysis could be possible. Using the formylated
locally minimized construct of GART, a high pK
a for His108
was calculated indicating a protonated histidine, and a low
pK
a for GAR(NH
a) was calculated indicating that
GAR is in neutral form. Our results are in qualitative agreement with
the current mechanistic picture of the catalytic process of GART,
deduced from the experimental data, but do not reproduce the absolute
magnitude of the pK
a's extracted from fits of
k
cat-pH profiles, possibly because the static time averaged
crystallogrphic structure does not describe adequately the dynamic
nature of the catalytic site during binding and catalysis. In addition,
a strong effect on the pK
a of GAR(NH
2) is produced
by the theoretical mutations of His108Ala and Asp144Ala, which is not in
agreement with the observed insensitivity of the pK
a of
GAR(NH
2) modeled from the experimental data, using similar
mutations. Finally, we show that important three-way electrostatic
interactions between highly conserved His137, with His108 and Asp144 are
responsible for stabilizing the electrostatic microenvironment of the
catalytic site. In conclusion, our data suggest that further detailed
computational and experimental work is necessary.
Kinetic Mechanism of End to End Annealing of Actin FilamentsErnesto Andrianantoandro, Laurent Blanchoin, David Sept, J. Andrew McCammon and Thomas D. PollardJournal of Molecular Biology, Vol. 312, Issue 4, pp. 721-730 (2001) [PubMed 11575927]We investigated the effect of actin filament length and capping protein
on the rate of end to end annealing of actin filaments. Long filaments
were fragmented by shearing and allowed to recover. Stabilizing
filaments with phalloidin in most experiments eliminated any
contribution of subunit dissociation and association to redistribution
of lengths but did not affect the results. Two different assays,
fluorescence microscopy to measure filament lengths and polymerization
to measure concentration of barbed filament ends, gave the same time
course of annealing. The rate of annealing declines with time as the
average filament length increases. Longer filaments also anneal slower
than short filaments. The second order annealing rate constant is
inversely proportional to mean polymer length with a value of 1.1
mM
-1s
-1/length in subunits. Capping protein slows
but does not prevent annealing. Annealing is a highly favorable reaction
with a strong influence on the length of polymers produced by
spontaneous polymerization and should be considered in thinking about
polymer dynamics in cells.