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Temperature dependent properties of a kinesin-3 motor
protein from Thermomyces lanuginosus
Susan B. Rivera a, Steven J. Koch a, Joseph M. Bauer b,
J. Matthew Edwards a, George D. Bachand a,*
a Biomolecular Interfaces and Systems Department, Sandia National Laboratories, P.O. Box 5800, MS-1413, Albuquerque, NM 87185-1413, USA
b Micro-Total-Analytical Systems Department, Sandia National Laboratories, P.O. Box 5800, MS-1425, Albuquerque, NM 87185-1425, USA
Received 4 January 2007; accepted 13 February 2007
Available online 21 February 2007
Abstract
Kinesins are cytoskeletal motor proteins that share a common mechanochemical motor domain, and are responsible for trafficking
macromolecules. Here we report the cloning and characterization of a monomeric, kinesin-3 (TKIN) from Thermomyces lanuginosus.
TKIN displayed a maximum rate of ATP hydrolysis at 55 C; the KATPm was also significantly greater at 50 C. Gliding motility rates
reached a maximum of 5.5 lm s1 at 45 C, which is among the highest rates reported for kinesin. Arrhenius energy barriers were cal-
culated to be 103 kJ mol1, nearly twofold greater than other mesophilic kinesin motors. The enthalpy of activation and entropy acti-
vation of TKIN were also significantly greater when compared to other mesophilic kinesins. A thermally induced aggregation of TKIN,
which could be moderated by the addition of ATP, was observed at temperatures above 45 C. Together, these results illustrate the
kinetic response and stability of this unique motor protein at elevated temperatures.
 2007 Elsevier Inc. All rights reserved.
Keywords: Motor proteins; Biomolecular motors; Kinesin; Microtubules; Thermophiles
1. Introduction
Kinesin motor proteins are diverse superfamily cytoskel-
etal transport proteins that are fundamental to a wide
array of cellular functions. In fungi, different classes of
kinesin are responsible for nuclear migration and position-
ing, spindle assembly during mitosis/meiosis, endosome
trafficking, secretion, vacuole organization, and mitochon-
dria positioning (Schoch et al., 2003; Steinberg, 1998;
Steinberg, 2000; Xiang and Plamann, 2003). Despite the
variety of functions in fungal cells, kinesins share common
structural similarities that include a motor domain, a neck-
linker region, and in many cases an a-helical coiled-coil tail
region. The biochemical and biophysical properties of con-
ventional kinesin (Kinesin-1 or KHC family) have been
well-characterized, and demonstrate the ability to effi-
ciently hydrolyze ATP while moving processively along
microtubule filaments in 8-nm steps using an asymmetric
hand-over-hand mechanism (Asbury et al., 2003; Sablin
and Fletterick, 2004; Schnitzer and Block, 1997; Yildiz
et al., 2004). The neck-linker region of KHC represents a
mechanical transducer for efficiently converting ATP
hydrolytic energy into mechanical movement (Vale and
Fletterick, 1997). The a-helical tail of KHC interacts to
form homodimers, and along with associated light chains,
is responsible for binding cargo (Howard, 1996).
Conventional kinesin from fungi display unique struc-
tural, biochemical, and biophysical properties relative to
kinesin from other eukaryotes. In general, the motility
velocity, processivity, and ATP hydrolysis rates of fungal
kinesins are significantly greater than those of eukaryotic
conventional kinesin (Grummt et al., 1998a; Steinberg
and Schliwa, 1995; Steinberg and Schliwa, 1996). These
differences have be attributed to distinct structural features
such as a helix disrupting hinge in the neck region, and a
1087-1845/$ - see front matter  2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2007.02.004
* Corresponding author. Fax: +1 505 844 5470.
E-mail address: gdbacha@sandia.gov (G.D. Bachand).
www.elsevier.com/locate/yfgbi
Available online at www.sciencedirect.com
Fungal Genetics and Biology 44 (2007) 1170–1179

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nucleotide-binding pocket that is more open (Grummt
et al., 1998b; Rice et al., 2003; Song et al., 2001). In addi-
tion, the ability of some fungal kinesins to interact with
both a and b tubulin, specifically the E-hooks, may also
contribute to the differences in catalytic properties (Grum-
mt et al., 1998b; Lakamper et al., 2003). While fungal
conventional kinesins apparently lack associated light
chains (Kirchner et al., 1999; Schoch et al., 2003; Steinberg,
1997; Steinberg and Schliwa, 1996; Steinberg et al., 1998), a
conserved tyrosine residue in the neck region of fungal
kinesin inhibits catalytic activity, mimicking the regulatory
functions of light chains found in other eukaryotic kinesins
(Schafer et al., 2003).
Although most members of the kinesin superfamily
form homo- and hetero-oligomers, members of the kine-
sin-3 (or KIF1/Unc104) family are primarily monomeric
(Bloom, 2001; Rogers et al., 2001), but have also been
shown to transition into a functional dimer state (Adio
et al., 2006; Al-Bassam et al., 2003; Rashid et al., 2005;
Tomishige et al., 2002). Kinesin-3 motors are involved in
the anterograde transport of synaptic vesicles in the neu-
rons (Lee et al., 2003; Okada et al., 1995), as well as the
transport of organelles such as the mitochondria and the
Golgi apparatus (Bloom, 2001; Schoch et al., 2003). In gen-
eral, the kinesin-3 motors display significantly faster
in vitro transport rates in multiple motor assays compared
with mammalian conventional kinesins (Okada et al., 1995;
Pierce et al., 1999). Despite the monomeric nature of these
motors, processive movement of KIF1A motors along
microtubule filaments has been reported, and attributed
to interaction of the K-loop with the microtubule (Nitta
et al., 2004; Okada and Hirokawa, 2000), a transition of
KIF1A in functional dimmers (Al-Bassam et al., 2003;
Tomishige et al., 2002), or by working in small teams
bound to a single cargo (Rogers et al., 2001).
Several kinesin-3 motor proteins have been isolated
from, or predicted from the sequenced genomes of ascomy-
cetous and basidiomycetous fungi (Adio et al., 2006; Fuchs
and Westermann, 2005; Sakowicz et al., 1999; Schoch
et al., 2003; Wedlich-Soldner et al., 2002). A kinesin-3
motor protein was isolated from the thermophilic, anamor-
phic fungus Thermomyces lanuginosus Tsiklinsky (Sak-
owicz et al., 1999). Initial characterization of this kinesin
demonstrated microtubule transport velocities of
2 lm s1 at room temperature, and the maintenance of
catalytic activity up to 45 C (Sakowicz et al., 1999). Simi-
larly, a monomeric kinesin related to the kinesin-3 family
was identified from the corn smut fungus Ustilago maydis
(DeCandolle), and shown to be responsible, along with
dynein, for transporting endosomes in fungal hyphae
(Wedlich-Soldner et al., 2002). Kinesin-3 motors, NKin2
and NKin3, facilitate mitochondria transport and position-
ing in Neurospora crassa Shear et Dodge (Fuchs and West-
ermann, 2005). Characterization of a recombinant NKin3
(or NcKin3) indicated that the motor was capable of form-
ing stable dimers, but moves along microtubules in a non-
processive manner (Adio et al., 2006). While the function
of kinesin-3 motor proteins in fungi has been studied, the
catalytic properties are poorly understood, particularly in
comparison their analogs from other eukaryotes. Thus,
the primary objective of our work was to attain a
fundamental understanding of the catalytic and tempera-
ture-dependent kinetic properties of a kinesin-3 motor
isolated from T. lanuginosus. We report the cloning and
expression of a monomeric, kinesin-3 motor protein from
T. lanuginosus, and the analysis of the temperature-depen-
dent kinetic properties and potential structural parameters
related to temperature of the recombinant enzyme.
2. Materials and methods
2.1. Construction and expression of recombinant TKIN
Thermomyces lanuginosus was obtained from the Amer-
ican Type Culture Collection (catalog # 36350), and grown
at 55 C in potato dextrose broth. cDNA was generated
from total RNA was isolated using SuperScript II reverse
transcriptase (Invitrogen, Carlsbad, CA) and oligo(dT)
primers, and subsequently amplified using sequence specific
primers (Forward: 5 0-ATGTCGGGCGGTGGAAATAT
CAA-3 0; Reverse: 5 0-GATATCGAATTCCTGCTTCG
CTG-3 0) based on the previously published sequence (Sak-
owicz et al., 1999). The resulting 2358-bp amplified product
was cloned into the pET-Blue1 expression system (Nova-
gen, Madison, WI), and a 10· poly-Histidine tag was
inserted at the 3 0 end of the amplified TKIN sequence. A
second clone of TKIN was also constructed to include a
thrombin recognition site (LVPRGS) immediately
upstream of the 10· poly-His tag, in order to evaluate
potential effects of the His-tag on kinesin functionality.
TKIN was expressed in Escherichia coli strain Tuner
(DE3) pLacI (Novagen, Madison, WI) grown in LB med-
ium with 100 lg mL1 ampicillin and 34 lg mL1 chloram-
phenicol, at 30 C with shaking at 250 rpm. Protein
expression was induced with 0.5 mM isopropyl-b-D-thioga-
lactoside when the OD600  0.7, and cells were harvested
by centrifugation 4 h after induction. Cell pellets were
resuspended in lysis buffer (50 mM phosphate buffer, pH
7.0, 500 mM NaCl, 30 mM imidazole, 5 mM bME, 1 mM
phenylmethylsulfonyl fluoride, and 100 lM ATP) and dis-
rupted with Bugbuster Protein Extraction Reagent (Nova-
gen, Madison, WI). The suspension was centrifuged at
40,000g for 20 min, and the supernatant was batch bound
to pre-equilibrated Ni2+–NTA resin (Qiagen, Valencia,
CA). The slurry was washed with a buffer consisting of
50 mM phosphate buffer, pH 7.0, 500 mM NaCl, 50 mM
imidazole, 100 lM ATP and 5 mM bME, and protein
was eluted with a buffer of 50 mM phosphate buffer, pH
7.0, 50 mM NaCl, 1 M imidazole, 100 lM ATP and
5 mM b-mercaptoethanol. Eluted fractions were analyzed
by SDS–PAGE; fractions containing purified TKIN were
combined, dialyzed against 20 mM phosphate buffer (pH
7.0), and precipitated with ammonium sulfate
(0.57 g mL1). The pellet protein was resuspended in
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BRB80 (80 mM Pipes, 1 mM MgCl2, 1 mM ethylene glycol
bis(b-aminoethyl ether) N,N 0-tetaacetic acid), and
exchanged into BRB80 or BRB80 with 0.1–10 mM ATP
using P6-spin columns (Bio-Rad Laboratories, Hercules,
CA). Protein concentrations were determined by the Brad-
ford method (Bradford, 1976). The native oligomeric state
of TKIN was estimated using size exclusion chromatogra-
phy on a Superdex 200 HR 10/300 GL (Amersham Biosci-
ences, Piscataway, NJ) gel filtration column equilibrated
with BRB80, 1 mM bME, 150 mM NaCl and 10 lM ATP.
2.2. Preparation of microtubules
Microtubules for kinetic analysis and gliding motility
assays were prepared by polymerizing unlabeled and rho-
damine-labeled tubulin (Cytoskeleton Inc., Denver, CO),
respectively, at a concentration of 5 mg mL1 in BRB80-
P (BRB80, 1 mM GTP and 10% glycerol) for 20 min at
37 C. The resulting microtubule solution was then stabi-
lized by the addition of BRB80-T (BRB80 with 10 lM
taxol), and diluted to the appropriate protein concentra-
tion. To determine the directionality of TKIN transport,
unmodified tubulin and N-ethylmaleimide modified
(NEM) tubulin, which inhibits minus end assembly (Phelps
and Walker, 2000), were used to polymerize taxol-stabi-
lized TRITC-labeled seeds, and subsequently microtubules
with an asymmetrically located bright rhodamine-labeled
minus end (Hyman et al., 1991).
2.3. Determining rate constants for ATP hydrolysis by TKIN
The kinetic rate constants for TKIN at ambient temper-
ature were evaluated by varying the concentration of ATP
at 25 C using a NADH-coupled double enzyme assay
(Huang and Hackney, 1994). To ensure that the poly-His
tag did not affect activity, ATPase rates of TKIN with
and without the poly-His tag were measured at room tem-
perature. Because no significant change in velocity was
observed, all subsequent assays were performed with the
His-tagged TKIN. To compare assay reproducibility, the
KATPm was also evaluated at ambient temperature using
the Malachite green assay described below. Two separate
experiments varying ATP concentrations from 0 to
1 mM, and 0 to 10 mM were preformed and results were
similar. The concentration of tubulin was determined by
absorbance at 275 nm in 6 M guanidine HCl using an
extinction coefficient of 1.03 mL mg1 cm, and expressed
in terms of 110 kDa tubulin heterodimers. The K50% MT
was determined by varying the tubulin concentration
between 0 and 2 lM. Kinetics rate constants and Michae-
lis–Menten plots were generated using Sigma Plot—
Enzyme Kinetics Module (SPSS Inc., Chicago, IL).
2.4. Temperature dependent ATP hydrolysis by TKIN
The ATPase hydrolytic activity of TKIN was measured
as a function of temperature using the Malachite Green
assay (Gilbert and Mackey, 2000). Solutions of BRB80-
T, 0.5 mM dithiothreitol, 7 mM ATP (based on the KATPm
results at 50 C), and 5.0 lM microtubules were pre-incu-
bated at specific test temperatures for 10 s before purified
TKIN (0.3–0.5 lM) was added to a final volume of
25 lL. Reactions were incubated for 120–300 s between
20 and 70 C (5 C increments), and then quenched with
2 M HCl. the phosphate concentration was determined
by adding a solution of 3 parts 0.045% malachite green
hydrochloride and one part 4.2% ammonium molybdate
in 4 N HCl, and reacting for 1 min. Reactions were
quenched by the addition of 3.5% sodium citrate, and the
absorbance at 660 nm was recorded. Data were corrected
for background signal using controls in which (i) TKIN
was not added to the reaction, and (ii) TKIN was added
to the reaction following quenching. Apparent Arrhenius
energy barriers were obtained by fitting an Arrhenius rela-
tionship to selected data points using non-linear fit in Sig-
maPlot (SysStat Software Inc., San Jose, CA).
2.5. Gliding motility assay using TKIN
A modification of the gliding motility assay (Howard
et al., 1993) was used to evaluate the microtubule transport
rate by TKIN. Briefly, a solution of 0.7 mg mL1 TKIN
(8 lM) in BRB80 and 100 lM ATP was infused a stan-
dard flow cell (i.e., a microscope slide, double-sided tape,
and a #1 glass coverslip), and allowed to incubate for
5 min. A motility solution, consisting of BRB80-T,
0.1–0.2 lM TRITC-microtubules, 6.25lM–10 mM MgATP,
and an oxygen scavenging system (Howard et al., 1993),
was then infused into the cell. Kinetic rate constants were
determined by varying ATP concentration in the motility
solution. Microtubule transport was observed at 100·
magnification using an Olympus BX51, and recorded using
a Hamamatsu C4742-98 CCD camera. A Bioptechs (But-
ler, PA) lens heater and a 6.5 cm2 Peltier heating device
were used to reliably control the fluid temperature in the
flow cell, and evaluate temperature-dependent changes in
motility velocities. Flow cells were sandwiched between
the two heating elements, which provided rapid and accu-
rate temperature cycling to within ±2 C. Time-lapse
images were processed using the MicroSuite software pack-
age (Olympus America, Inc., Hauppauge, NY) to estimate
transport velocities.
2.6. Temperature-dependent aggregation of TKIN
Characterization of TLKIF1 suggested that enzymatic
activity was lost following incubation above 45 C
(Sakowicz et al., 1999). Thus, to further evaluate the tem-
perature-dependent states of TKIN, samples of purified
TKIN (3 mg mL1) were heated for 3 min at 10 C inter-
vals between 30 and 80 C, and allowed to cool to room
temperature. Glutaraldehyde was then added to a final
concentration of 90 lM, and incubated for 1 h at room
temperature. The extent of aggregation was analyzed using
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SDS–PAGE. The transition temperature was further
verified by incubating 1 mg mL1 samples of TKIN
between 38 and 45 C (1 C intervals) for 5 min, and ana-
lyzing aggregation by SDS–PAGE.
Temperature-dependent changes in the aggregation of
TKIN were also monitored using a DynaPro-LSR (Pro-
terion Corp., Piscataway, NJ) temperature controlled
dynamic light scattering system. The 90-degree scattering
intensity (related to protein particle sizes) was monitored
in 5-s intervals and the data were software filtered (Dynam-
ics V5, Proterion Corp.) excluding data outside a baseline
range of 0.99–1.01. In order to identify the transition tem-
perature for rapid aggregation, a 0.2 mg mL1 solution of
TKIN in BRB80 was stepped between 5 and 45 C, and
back to 5 C. Further, the effect of ATP on heat-induced
aggregation was evaluated by preparing separate samples
of 0.35 mg mL1 TKIN in BRB80 supplemented with 0,
1, 10, 100, or 1000 lM ATP, and monitoring the scattering
intensity over time following a temperature increase of
25–45 C.
3. Results
3.1. TKIN sequencing and structural characterization
Analysis of the TKIN protein sequence (GenBank
Accession No. AY623608) using the SMART program
(http://smart.embl-heidelberg.de) predicted structural fea-
tures characteristic of the kinesin-3 subfamily of kinesins
(Hirokawa and Takemura, 2005; Schoch et al., 2003). A
kinesin motor domain was predicted between amino acids
4–367 (E-value = 3.66 · 10177), and showed significant
homology to other fungal kinesin-3 motor proteins
(Fig. 1b). TKIN also contained a forkhead associated
(FHA) (amino acids 517–569, E-value = 1.36 · 102)
domain, which is common to kinesin-3 motors and believed
to interact with phosphopeptides (Fuchs and Westermann,
2005). Two coiled coil regions, CC1 and CC2 (amino acids
436–477, and 755–786), flanking in the FHA domain were
also predicted, consistent with other kinesin-3 motors.
Based on the primers using for amplification, the TKIN
construct did not contain the pleckstrin homology (PH)
domain that is common to most kinesin-3 motors. This
domain plays a central role in the interaction of kinesin-3
with lipids and lipid rafts of membrane cargo (Klopfenstein
et al., 2002), and does not affect the catalytic properties of
the enzyme. Additionally, only three lysine residues were
present between a4 and a5 in TKIN, compared with six
lysines in KIF1A (Fig. 1a). This lysine-rich domain is
believed to bind to the negatively charged regions of the
microtubule, and regulate the processivity of monomeric
KIF1A (Okada and Hirokawa, 2000). The low number
of lysines in the TKIN domain is unlikely to promote pro-
cessivity based on an insufficient density of positive charge
(Bloom, 2001).
Phylogenetic analysis of TKIN (Fig. 1b) indicates that it
belongs to the KIN2/KLP8 sub-group of kinesin-3 motor
proteins (Fuchs and Westermann, 2005), with KIN2 from
Aspergillus nidulans (Eidam) Winter as the closest neigh-
bor. A comparison of TKIN with a previously cloned
TLKIF1 protein from T. lanuginosus (Sakowicz et al.,
1999) displayed a number of differences in both the DNA
and deduced amino acid sequences (Fig. 1c), suggesting
that it may be an isoform. The first region (amino acids
423–427) replaced a Gln residue with an Arg, and an
Ala with a Gly (Fig. 1b). In region two (amino acids
487–493), a shift in the reading frame resulted in five amino
acid changes, including changes in charge (Glu to Lys, and
His to Thr) and aromaticity (Tyr to Thr, Fig. 1b). In the
last region (amino acids 747–748), two uncharged, hydro-
phobic Phe residues were replaced by Lys residues. In all
cases, these observed differences occurred adjacent to the
coiled coil domains within TKIN.
3.2. TKIN ATP hydrolysis and motility properties
Recombinant TKIN was purified (>95%) by Ni2+–NTA
affinity chromatography, and observed to have a molecular
weight of 90 kDa as determined by SDS–PAGE and size
exclusion chromatography (not shown), consistent with the
calculated molecular weight of monomeric TKIN
(88,678 Da). The observed 90-kDa peak in size exclusion
chromatography suggests that TKIN exists as a monomer
at room temperature.
The ATP hydrolytic properties of TKIN were evaluated
at ambient temperature using both the NADH-coupled
double enzyme (Huang and Hackney, 1994) and the Mala-
chite Green (Gilbert and Mackey, 2000) assays. The in vitro
motility properties were characterized with the gliding
motility assay (Howard et al., 1993). The ATP hydrolytic
activity displayed typical Michaelis–Menten kinetics for
both substrates, ATP and microtubules, at 25 C as shown
in Fig. 2a. Removal of the poly-His tag from TKIN by
thrombin digestion did not significantly change the kinetic
constants obtained at room temperature. The microtubule
concentration required for half-maximal velocity (i.e., K50%
MT) was 0.34 ± 0.09 lM at 25 C, comparable to that
observed for KIF1D in similar ionic strength buffers (Rog-
ers et al., 2001), and a truncated construct of Unc104
(Al-Bassam et al., 2003). The KATPm value of 42 ± 6.8 lM
was determined at 25 C, and similar to that observed for
other kinesin-3 motor proteins (Rogers et al., 2001; Wed-
lich-Soldner et al., 2002) and conventional kinesins (Bo¨hm
et al., 2000a).
TKIN supported microtubule gliding in gliding motility
assays with velocities of 1.5 lm s1 at 25 C, which is
consistent with TLKIF1 rates (Sakowicz et al., 1999) and
other kinesin-3 motors (Rogers et al., 2001). The minus-
end of the microtubule was the leading end in gliding
assays using asymmetrically labeled microtubules, confirm-
ing that TKIN is a plus-end directed motor. Microtubule
gliding was observed for TKIN concentrations as low as
0.1 mg mL1, although the actual fraction of active pro-
teins bound to the surface was not determined. While the
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gliding rate did not change measurably as a function of
motor concentration, the number of microtubules bound
to the surface decreased with decreasing kinesin concentra-
tion. The gliding rates of TKIN were studied with respect
to ATP concentration and kinesin concentration. As
expected, gliding rates increased as a function of ATP con-
centration, and followed standard Michaelis–Menten
kinetics (Fig. 2b). A KATPm of 38.5 ± 8.7 lM was deter-
mined based on the gliding motility data, and consistent
with that determined for ATP hydrolysis, within experi-
mental error.
3.3. Temperature dependent ATP hydrolysis and motility
The temperature dependent properties of TKIN were
evaluated in both ATP hydrolysis and gliding motility
assays. The measured rate of ATP hydrolysis between 20
and 40 C was relatively constant (1 s1; Fig. 3a). This
observation may be attributed to either (1) turnover rates
below the signal-to-noise threshold of the assay, or (2) a
distinct transition in TKIN structure at 45–50 C that
induces increased ATP hydrolysis. We feel the first expla-
nation is most likely, since gliding motility assays showed
Arrhenius behavior at these temperatures. A distinct break
in the Arrhenius plots was previously observed, however
for porcine kinesin at 27 C, and attributed to partial melt-
ing in the N-terminal portion of the a-helix, lowering the
activation energy (Bo¨hm et al., 2000). Thus, a structural
transition in TKIN at 45–50 C resulting in a lower activa-
tion energy cannot be precluded. The ATP hydrolysis rate
did display a sharp increase between 45 and 65 C (Fig. 3a),
and displayed microtubule-based activation across this
range. The hydrolytic activity of TKIN displayed a clear
temperature dependency with an observed maximum at
60–65 C. The KATPm value was determined at 50 C, and
displayed a significant increase (1600 ± 300 lM) as com-
pared to that observed at 25 C.
The gliding velocity also displayed a clear temperature
dependency with a maximum velocity of 5.5 lm s1 at
45 C, which is among the highest rates reported for any
kinesin isolated to date. Microtubules failed to bind to
the surface-bound kinesin at temperatures above 50 C,
Fig. 1. (a) Structure of TKIN with the L12 region expanded to show the K-loop of Mus musculus, and its apparent absence from TKIN. (b) Homology
tree of representative kinesin family members, showing a close relationship between TKIN and members of the KIN2/KLP8 sub-group. The tree includes
Kinesin-3 motors from Aspergillus nidulans (genome annotation No. AN7547.2 and AN6863.2), Botryotinia fuckeliana (GenBank Accession No.
AAO59283 and AAO59284), Gibberella moniliformis (GenBank Accession No. AAO59306), G. zeae (GenBank Accession No. XP390365), Neurospora
crassa (Genome Annotation No. NCU06733.1 and NCU03715.1), Magnaporthe grisea (Genome Annotation No. MG09255.4), Cochliobolus
heterostrophus (GenBank Accession No. AAO59294 and AAO59295), Ustilago maydis (GenBank Accession No. AAL87137), Drosophila melanogaster
(GenBank Accession No. X99617), Caenorhabditis elegans (GenBank Accession No. M58582), and Homo sapiens (GenBank Accession No. AF279865).
Conventional kinesin from D. melanogaster (GenBank Accession No. M24441), M. musculus (GenBank Accession # U86090), and N. crassa (GenBank
Accession No. L47106) were also included. (c) Comparison of the amino acid sequences of TKIN and the previously reported kinesin TLKIF1 (Sakowicz
et al., 1999).
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and quickly depolymerized after approximately 30 s likely
due to both temperature and photo-induced oxidation of
the fluorescent tubulin.
Arrhenius energy barriers (Ea) were estimated based on
the data for both the ATP hydrolysis and gliding motility
assays. Only selected data were used to estimate these val-
ues due to (1) the lack of temperature dependent changes in
ATP hydrolysis observed at lower temperatures, and (2)
problems with microtubule depolymerization at higher
temperatures (e.g., >60 C). The Ea values were estimated
to be 112 ± 11 kJ mol1 (26.8 ± 2.7 kcal mol1; P < 0.01)
and 94 ± 9 kJ mol1 (22.4 ± 2.1 kcal mol1; P < 0.01) based
on the ATP hydrolytic and gliding motility data, respec-
tively (Fig. 3b). The Arrhenius energy barrier and kinetic
data were used to calculate the enthalpy of activation
(DH), the entropy activation (TDS), and the free energy
of activation (DG) for TKIN. These values were also cal-
culated using the Arrhenius energy barriers and kinetic
data for Eg5 (Crevel et al., 1997), ncd (Crevel et al.,
1997), and bovine kinesin (Kawaguchi and Ishiwata,
2000) as a reference. TKIN displayed an increased DH
and TDS with respect to the other kinesins (Table 1).
Interestingly, there was no observed difference in the free
energy of activation, which is also expected to change
due to the thermostable nature of TKIN.
3.4. Temperature-dependent aggregation of TKIN
The previous report (Sakowicz et al., 1999) indicated
that TLKIF1 lost hydrolytic activity at 40–45 C, which
contrasts with the ATP hydrolysis and gliding motility data
for TKIN (Fig. 3). The thermal stability of TKIN was fur-
ther examined by dynamic light scattering (DLS) and SDS–
PAGE analysis to understand this discrepancy. Solutions
of TKIN formed a viscous and translucent physical gel
between 40 and 50 C; SDS–PAGE (not shown) results dis-
played the formation of high molecular weight aggregates
at temperatures above 45 C. Removal of the His-tag from
TKIN by thrombin digestion did not affect the observed
temperature-induced aggregation, thus precluding the
involvement of His-tag in the observed aggregation phe-
nomenon. Similarly, extensive protein aggregation at ele-
vated temperatures was observed using dynamic light
scattering (DLS; Fig. 4a). A persistent increased scattering
intensity following reduction in the solution temperature
suggests that the aggregation process is irreversible. These
Fig. 3. (a) Temperature dependent effects on bulk specific activity (-d-)
and in vitro motility (-s-) of TKIN. Malachite green assays were used to
measure the turnover of TKIN at temperatures ranging from 20 to 70 C.
(b) Plots of ln(rate) vs. 1/T of selected ATP hydrolysis (R2 = 0.96) and
motility (R2 = 0.97) data show that the Arrhenius energy barrier is nearly
twice as high as reported for other kinesin (Bo¨hm et al., 2000; Kawaguchi
and Ishiwata, 2000). Error bars represent the standard error for bulk
measurements. Data represent a subset of the values in (a) as described in
text.
Fig. 2. (a) Representative plots of Michaelis–Menten kinetics at 25 C for
ATP (-s-) and microtubules (-d-) for ATP hydrolysis. The KATPm was
42 ± 6.8 lM and significantly increased to 1600 ± 300 lM at 50 C. (b)
Michaelis–Menten kinetics at 25 C for ATP determine by gliding motility
assays; the KATPm was 38.5 ± 8.7 lM.
S.B. Rivera et al. / Fungal Genetics and Biology 44 (2007) 1170–1179 1175

Author's personal copy
data suggest that TKIN undergoes a heat-induced aggrega-
tion, and likely explain the drop in activity observed for
TLFKIF1 (Sakowicz et al., 1999) after pre-incubation at
45 C.
To further investigate this aggregation phenomenon, the
role of ATP and buffer components was evaluated using
DLS. The role of microtubules in limiting aggregation
could not be determined because polymerized tubulin dom-
inates the light scattering signal. Heat-induced aggregation
was significantly reduced by the addition of ATP (Fig. 4b),
indicating it plays a role in stabilizing TKIN. Heat-induced
aggregation was not affected by addition of 300 mM NaCl,
7 mM MgCl2, 5 mM b-ME, 0.25% Tween 20, or changing
the Pipes buffer counterion from Na+ to K+ (not shown).
Given the insensitivity of heat-induced aggregation to these
solutes and the strong dependence on only moderate con-
centrations of ATP, it appears that ATP could play an
important and specific role in aggregation of TKIN, which
should be considered for storage and future in vitro
applications.
4. Discussion
4.1. Structural and chemical features of TKIN
This study demonstrates that TKIN represents a unique,
thermostable member of the kinesin-3 (KIF1/Unc104)
family. Phylogenetic analysis suggest that TKIN is a mem-
ber of the KIN2/KLP8 sub-group (Fig. 1b); members of
this group are responsible for mitochondrial movement
(Fuchs and Westermann, 2005). KIN2 from Aspergillus
nidulans (Eidam) Winter is the closest neighbor, a trend
reported for other proteins from T. lanuginosus and
Aspergillus spp. (Hakulinen et al., 2003). TKIN displayed
distinct sequence differences with TLKIF1 (Sakowicz
et al., 1999). In particular, a nucleotide deletion at 1460
resulted in a shift in the reading frame that corresponded
to a change in five amino acids between the CC1 and
FHA domains (Fig. 1b); a nucleotide insertion at 1758
restored the reading frame. While the differences resulted
in minor changes in charge and hydrophobicity, the basic
catalytic properties of TKIN were similar to those reported
for TLKIF1 (Sakowicz et al., 1999).
Members of the kinesin-3 family display a range of func-
tional oligomeric states. The type member of this family
(i.e., KIF1A) is capable of moving processively as an inde-
pendent monomer (Nitta et al., 2004; Okada and Hiroka-
wa, 1999; Okada and Hirokawa, 2000), and can also
work synergistically as a functional dimer (Al-Bassam
et al., 2003; Rashid et al., 2005; Tomishige et al., 2002).
The oligomeric state of fungal kinesin-3 motor proteins
has not been previously studied. Our data suggest that
TKIN is primarily monomeric. While TKIN is a truncated
construct, it but does contain the structural features (e.g.,
Table 1
Comparison of the thermodynamic properties of TKIN, Eg5, ncd, and bovine kinesin
Kinesin type Assay type Ea (kJ mol1) DG (kJ mol1) DH (kJ mol1) TDS (kJ mol1)
TKIN ATPase 112 ± 11b 76 109 33
TKIN Motility 94 ± 9 73 91 19
Eg5a Motility 61 79 59 20
ncda Motility 63 78 61 17
Bovine kinesina Motility 50 74 48 26
a Values calculated for Eg5 and ncd based on Kcat and Ea values from Crevel et al. (1997), and bovine kinesin based on Kcat and Ea values from
Kawaguchi and Ishiwata (2000).
b Standard error of the mean.
Fig. 4. (a) Results of temperature controlled dynamic light scattering
experiments that indicate that TKIN irreversibly aggregates at 45 C.
Open circles represent the scattering intensity (megacounts s1) and solid
squares represent the sample temperature. The vertical dashed line
indicates the time that the sample was raised to 45 C. (b) Aggregation
of TKIN is suppressed by moderate concentrations of ATP. Curves show
dynamic light scattering intensity of a 4 lM TKIN sample held at 45 C,
with the concentration of ATP shown next to end of traces. Increasing
scattering signal is indicative of increasing aggregate sizes.
1176 S.B. Rivera et al. / Fungal Genetics and Biology 44 (2007) 1170–1179

Author's personal copy
neck coiled-coil) involved in monomer-dimer transition
(Adio et al., 2006; Al-Bassam et al., 2003; Tomishige
et al., 2002). Size exclusion chromatography, however,
demonstrated only a single peak (not shown) consistent
with the predicted molecular weight for the TKIN mono-
mer. Further investigation of full-length constructs of
TKIN will be necessary to fully determine the native olig-
omeric state of TKIN.
TKIN supports microtubule gliding velocities consistent
other members of the kinesin-3 subfamily (Okada and
Hirokawa, 1999; Sakowicz et al., 1999; Wedlich-Soldner
et al., 2002). This result contrasts the significant differences
in the motility velocity reported for conventional kinesin
(kinesin-1) isolated from fungi as compared to their mam-
malian analogs (Steinberg, 1997; Steinberg and Schliwa,
1995; Steinberg and Schliwa, 1996). Despite the high veloc-
ities observed in gliding motility assays, the turnover rate
for TKIN was relatively low across all temperatures. This
observation also confirms similar findings previously for
TLKIF1 (Sakowicz et al., 1999). The discrepancy between
the fast velocity and turnover rates may be explained if
TKIN is not processive, and a collection of motors is
responsible for propelling a single microtubule (Spudich,
1990). This contention that TKIN is a chemically non-pro-
cessive, monomeric kinesin-3 motor is further supported by
our sequence and chemical data. Based on the deduced
amino acid sequence of Loop 12, TKIN likely lacks a suf-
ficient charge density to promote processive movement
based on the biased Brownian-movement model (Okada
and Hirokawa, 2000). It has been hypothesized that this
domain binds to the negatively charged regions of the
microtubule to enable the processivity of monomeric
KIF1A (Okada and Hirokawa, 2000). Because TKIN con-
tains only three lysine residues in this domain (Fig. 1a), the
density of positive charge is unlikely to support processive
movement. Other fungal kinesin-3 motors such as NcKin3
have also been shown to possess a less charged K-loop
region and function in a non-processive manner (Adio
et al., 2006). TKIN also displayed a low ATPase turnover
rate compared with other kinesin (Okada and Hirokawa,
1999; Pierce et al., 1999), but similar to other non-proces-
sive kinesin such as KIF1D (Rogers et al., 2001). Thus,
our data support the notion that TKIN is a monomeric,
non-processive member of the kinesin-3 family. It has been
proposed that non-processive motors may function in
‘‘small teams’’ as a means of achieving organelles transport
over long distances (Rogers et al., 2001). Such a mechanism
could apply to TKIN as well as other non-processive motor
proteins with respect to trafficking organelles in fungal
hyphae.
4.2. Temperature-dependent properties of TKIN
TKIN was capable of supporting ATP hydrolysis at
temperatures higher than that reported for other kinesins
(Bo¨hm et al., 2000; Crevel et al., 1997; Kawaguchi and
Ishiwata, 2000). The rate of ATP hydrolysis peaked at
55 C, which is consistent with the optimal growth tem-
perature for T. lanuginosus. TKIN displayed typical
Michaelis–Menten kinetics at 25 and 50 C; a significant
increase in the KATPm , however, was observed at 50 C for
TKIN. Substantial increases in the Km at elevated temper-
atures have been reported for other thermophilic enzymes
(Georlette et al., 2000), but not for any kinesins. A small,
temperature-dependent change in the KATPm was previously
reported for porcine conventional kinesin, but was evalu-
ated over a much smaller temperature range (Bo¨hm
et al., 2000). A lack of temperature-dependent change in
ATP hydrolysis rate of TKIN at lower temperatures (i.e.,
20–40 C) was observed, and attributed to either a struc-
tural transition in TKIN or hydrolysis rates below the sig-
nal-to-noise threshold. A structural change in the a-helix of
porcine kinesin has been shown to lower the activation
energy, resulting in a distinct break in the Arrhenius plot
(Bo¨hm et al., 2000). Similar conformation changes in
enzyme structure have also been shown to result in discon-
tinuous Arrhenius plots (Singleton and Amelunxen, 1973).
Thus, a temperature-dependent structural change in TKIN
could explain the distinct responses in the rates of ATP
hydrolysis observed below and above 50 C.
TKIN supported microtubule gliding at elevated tem-
peratures, and displayed a maximum rate of 5.5 lm s1
at 45 C, which is among the highest rates reported for
any kinesin isolated to date. The gliding motility rates dis-
played a relatively linear response as a function of temper-
ature (Fig. 3a), unlike the ATP hydrolysis plot in which
two distinct responses were noted. Adhesion of TKIN to
the glass surface may have prevented any potential confor-
mational changes corresponding to a change in enzyme
function, as suggested for ATP hydrolysis. Microtubules
quickly detached from TKIN and depolymerized at tem-
peratures of 50 C and above. Thermal instability of bovine
tubulin and increased rates of photo-oxidation are likely
causes for these observations (Boal et al., 2006; Brunner
et al., 2004). Thermal-tolerant microtubules assembled
with tubulin isolated from T. lanuginosus would provide
more reliable data at higher temperatures.
The thermodynamic properties of TKIN were consistent
with other thermophilic enzymes. The Arrhenius energy for
TKIN was estimated at 103 kJ mol1 (24.5 kcal mol1),
which is nearly twofold greater than those reported for
conventional and ncd-like kinesin (Bo¨hm et al., 2000; Kaw-
aguchi and Ishiwata, 2000). Similarly, TKIN displayed an
increased DH and TDS as compared with other kinesins.
While these kinesins belong to different families, thermo-
stable enzymes generally display increased Arrhenius
energy barriers, as well as increased values for DH and
TDS compared with mesophilic analogs (Collins et al.,
2003).
4.3. Heat-induced aggregation of TKIN
Incubation of TKIN at 45 C and above resulted in the
formation of a viscous gel due to heat-induced aggregation
S.B. Rivera et al. / Fungal Genetics and Biology 44 (2007) 1170–1179 1177

Author's personal copy
of the enzyme. Intuitively, the aggregation of TKIN
observed at 45 C should not be caused by thermal
denaturation, as the optimum growth temperature of
T. lanuginosus is 55–60 C. Because aggregation was not
observed in ATP hydrolytic assays in which both microtu-
bules and ATP were present, it was hypothesized that the
aggregation phenomenon was substrate-mediated. DLS
experiments confirmed that the heat-induced aggregation
could be mediated, but not reversed, by the addition of
ATP at concentrations as low at 10 lM. Although microtu-
bules also could play an important role in mediating this
phenomenon, their role could not be discerned due to the
intrinsic high scattering associated with microtubules in
these assays. These data suggest a fundamental role of
ATP in maintaining the structural integrity of TKIN,
which is important for in vitro stability.
It was previously reported that the TLKIF1 kinesin
from T. lanuginosus lost enzymatic activity at 45 C, which
was attributed to either truncation/modification of the
expressed enzyme, or lack of a host-mediated stabilization
mechanism (Sakowicz et al., 1999). This observation is con-
sistent with the heat-induced aggregation observed for
TKIN at the same temperature. Thus, heat-induced aggre-
gation of TLKIF1 likely explains the observed loss of
hydrolytic activity, if ATP was not present during the incu-
bation step. Interesting, the heat-induced aggregation phe-
nomenon occurs in the temperature range in which TKIN
also begins displaying temperature-dependent changes in
the ATP hydrolytic rate. This observation further supports
the putative role of structural changes in regulating ATP
hydrolysis as discussed above. Further investigation, how-
ever, is necessary to fully discern the role of structural
changes with respect to both the heat-induced aggregation
phenomenon and ATP hydrolytic properties of TKIN.
Acknowledgments
We thank Amanda Trent for help in understanding the
heat-induced aggregation of TKIN. This work was par-
tially supported by Sandia’s Laboratory Directed Research
and Development Office, and the Defense Science Office of
the Defense Advanced Research Projects Agency. Sandia is
a multiprogram laboratory operated by Sandia Corpora-
tion, a Lockheed Martin Company, for the United States
Department of Energy’s National Nuclear Security Admin-
istration under Contract DE-AC04-94AL85000.
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