From the Department of Pharmaceutical Sciences,
School of Pharmacy, the § Department Biochemistry and
Molecular Genetics, and the ¶ Molecular Biology Program, School of
Medicine, University of Colorado Health Sciences Center, Denver,
Colorado 80262
Received for publication, January 18, 2001, and in revised form, February 21, 2001
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ABSTRACT |
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Terminase enzymes are common to double-stranded
DNA viruses. These enzymes "package" the viral genome into a
pre-formed capsid. Terminase from bacteriophage Terminase enzymes are common to many of the double-stranded
DNA bacteriophage and eukaryotic DNA viruses such as adenovirus and the
herpesvirus groups (2-4). These enzymes function to insert a viral
genome into the confines of a preformed, empty capsid. The terminase
enzyme from bacteriophage Replication of The gpA subunit of Our laboratory is interested in the biochemical and biophysical
mechanisms of DNA packaging by phage We recently described the construction and characterization of two
deletion mutants of gpNu1, gpNu1 Here we present a detailed biophysical characterization of
gpNu1 Materials and Methods--
Tryptone, yeast extract, and agar
were purchased from DIFCO. Restriction enzymes were purchased from
Promega. DEAE-Sepharose FF and SP-Sepharose FF chromatography resins
were purchased from Amersham Pharmacia Biotech. Restriction enzymes
were purchased from Promega. Guanidinium hydrochloride was purchased
from Mallinckrodt. All other materials were of the highest quality
commercially available.
Bacterial cultures were grown in shaker flasks utilizing a New
Brunswick Scientific series 25 incubator-shaker. All protein purifications utilized a Amersham Pharmacia Biotech fast-protein liquid
chromatography system that consisted of two P500 pumps, a GP250-plus
controller, a V7 injector, and a Uvicord SII variable-wavelength detector. UV-visible absorbance spectra were recorded on a
Hewlett-Packard HP8452A spectrophotometer. Fluorescence spectra were
recorded at room temperature on a PTI QuantaMaster spectrofluorometer. A protein concentration of 10 µg/ml in 10 mM potassium
phosphate buffer, pH 7.4, was used, and a buffer blank was subtracted
from the fluorescence spectrum. Circular dichroism (CD) spectra were recorded on an Aviv model 62DS circular dichroism spectropolarimeter equipped with a Brinkmann Lauda RM6 circulating water bath and a
thermostated cell holder. Near-UV CD spectra utilized a protein concentration of 1 mg/ml in a 0.1-cm strain-free cuvette. Data were
typically collected between 250 and 350 nm at 0.5-nm intervals using a
bandwidth of 1.5 nm and a dwell time of 30 s. Far-UV CD spectra
utilized a protein concentration of 100 µg/ml in a 0.1-cm strain-free
cuvette. Data were typically collected from 180 to 260 nm at 0.5-nm
intervals using a bandwidth of 1.5 nm and a dwell time of 30 s.
The raw spectra were converted to molar ellipticity using,
Bacterial Strains, DNA Preparation, and Protein
Purification--
Escherichia coli BL21(DE3) cells were a
generous gift of D. Kroll (University of Colorado Health Sciences
Center, Denver, CO). All synthetic oligonucleotides used in this study
were purchased from Life Technologies, Inc. and were used without
further purification. Plasmids pSF1 and pAFP1, kindly provided by M. Feiss (University of Iowa, Iowa City, IA), were purified from the
E. coli cell lines C600[pSF1] and JM107[pAFP1],
respectively, using Qiagen DNA prep columns. All of our purified
proteins were homogenous as determined by SDS-PAGE and densitometric
analysis using a Molecular Dynamics laser densitometer and the
ImageQuaNT data analysis package. Unless otherwise indicated, protein
concentrations were determined spectrally using millimolar extinction
coefficients (1, 32).
Construction of pNu1 Expression and Purification of gpNu1
The ammonium sulfate pellet was taken into buffer A and, after dialysis
against the same buffer, loaded onto a DEAE-Sepharose column (200 ml)
also equilibrated with buffer A. The column was developed with a salt
gradient with gpNu1 Sedimentation Equilibrium Analysis--
Experiments were
carried out with a Beckman XL-A analytical ultracentrifuge equipped
with a Ti-60 four-hole rotor with six-channel, 12-mm path-length
centerpieces. Absorbance optics were used throughout. Three different
protein concentrations were used with ratios of 10:3:1, with the
highest protein concentrations of 150 µM (
Models incorporating different assembly stoichiometries were based upon
the general equation,
Thermal Stability Studies--
Thermally induced protein
denaturation experiments were performed as described previously (32,
33, 35). Each data set represents the average of at least two
independent experiments. The fraction of protein in the denatured state
(FD) was determined using,
We have previously described the construction,
expression, and biochemical characterization of gpNu1 Thermally Induced Unfolding of gpNu1 Limited Proteolysis of gpNu1 Characterization of gpNu1
The UV absorbance spectrum of gpNu1
The far-UV CD spectrum of gpNu1 Stoichiometry of the Self-assembly Reaction--
The assembly
state of gpNu1 Thermally Induced Unfolding of gpNu1 GpNu1, the small terminase subunit, is responsible for
site-specific assembly of a holoenzyme complex required for genome packaging. The protein is further responsible for the exceptional stability of multiple nucleoprotein intermediates along the packaging pathway. Insolubility of the isolated subunit has hampered biochemical analysis of the protein. We have previously described the construction and biochemical characterization of gpNu1 Fluorescence, CD, and solution NMR studies demonstrated significant
secondary and tertiary structure in the construct (1). Thermally
induced unfolding of gpNu1 is composed of gpA
(72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the
expression and biochemical characterization of gpNu1
K100, a
construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q.,
de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and
Catalano, C. E. (1999) Biochemistry 38, 465-477).
Here we present a biophysical characterization of this construct.
Thermally induced loss of secondary and tertiary structures is fully
reversible. Surprisingly, although loss of tertiary structure is
cooperative, loss of secondary structure is non-cooperative. NMR and
limited proteolysis data suggest that
30 amino acids of gpNu1
K100
are solvent-exposed and highly flexible. We therefore constructed
gpNu1
E68, a protein consisting of the N-terminal 68 residues of
gpNu1. gpNu1
E68 is a dimer with no evidence of dissociation or
further aggregation. Thermally induced unfolding of gpNu1
E68 is
reversible, with concomitant loss of both secondary and tertiary
structure. The melting temperature increases with increasing protein
concentration, suggesting that dimerization and folding are, at least
in part, coupled. The data suggest that gpNu1
E68 represents the
minimal DNA binding domain of gpNu1. We further suggest that the
C-terminal
30 residues in gpNu1
K100 adopt a pseudo-stable
-helix that extends from the folded core of the protein. A model
describing the role of this helix in the assembly of the packaging
apparatus is discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is composed of two virally encoded
proteins, gpNu1 (181 amino acids) and gpA (640 amino acids), in a
gpA1·gpNu12 holoenzyme complex (5, 6).
Terminase holoenzyme possesses site-specific nuclease (6-9), ATPase
(10-13), DNA strand separation (9, 14), and DNA translocase (15, 16)
catalytic activities that work in concert to package viral DNA. All of
the terminase enzymes characterized to date possess a similar
holoenzyme composition (small and large subunits) and catalytic
activities (5, 17-21).
DNA proceeds through a rolling circle mechanism that
gives rise to linear concatemers of the viral genome linked in a head
to tail fashion (22, 23). Packaging of viral DNA requires the excision
of an individual genome from the concatemer, and packaging of the
48.5-kb1 duplex within the
capsid. Genome packaging by
terminase has been described in detail
(21, 24-27) and is summarized here. Packaging initiates with the
assembly of the holoenzyme at a cos site in the concatemer.
This site represents the junction between the left and right ends of
individual genomes within the concatemer (Fig. 1A).
Site-specific assembly at cos is mediated by cooperative gpNu1 binding to three repeated R-elements in the cosB
subsite of cos. Assembly of gpNu1 at cosB
promotes the assembly of a gpA dimer symmetrically disposed at
cosN, yielding a stable pre-nicking complex. Site-specific
nicking of the duplex at cosN, followed by an
ATP-dependent separation of the nicked strands, yields
complex I, the next stable intermediate. This nucleoprotein complex
next binds an empty capsid, which triggers the transition to a mobile, ATP-driven translocation complex that inserts DNA into the capsid. Upon
arrival at the next downstream cos site, terminase again nicks the duplex, and strand separation results in release of the
DNA-filled capsid and re-generation of complex I.
terminase appears to possess all of the
catalytic activities required for genome packaging, but the efficiency of each reaction is strongly stimulated by the smaller gpNu1 subunit (7, 8, 12-14, 16, 28). Moreover, gpNu1 is required for specific and
high affinity gpA DNA binding interactions (29) and likely contributes
to the exceptional stability of the pre-nicking complex and complex I
(30, 31).
terminase. Central to the
packaging process is the cooperative assembly of gpNu1 and gpA at
cos (Fig. 1A). To define this assembly process at
a molecular level requires an understanding of the structural features governing physical interaction between the enzyme subunits and with
DNA. Toward this end, we have sought to define the properties governing
intrinsic and cooperative DNA binding by gpNu1. Unfortunately, the
isolated gpNu1 subunit shows a strong tendency to aggregate upon
concentration (9, 28, 32-34), a feature that has hampered structural
and biophysical characterization of the protein.
P141 (35) and gpNu1
K100 (1),
which are proteins truncated at Pro141 and
Lys100 of full-length gpNu1, respectively. Studies of these
constructs led to a model where the C-terminal 40 residues of the
protein are required for interactions with the gpA subunit to form a
catalytically competent holoenzyme complex (Fig. 1B).
Residues 100-140 promote self-association interactions that mediate
cooperative DNA binding. The N-terminal 100 residues of the protein
represented by gpNu1
K100 contain the putative helix-turn-helix DNA
binding motif postulated to play a direct role in DNA binding. Indeed,
the construct is folded in solution and binds cos-containing
DNA with reasonable specificity (1). Preliminary NMR experiments
suggested that gpNu1
K100 would be amenable to structural studies
(1).
K100. These studies suggest that the construct consists of a
functional N-terminal domain that possess a pseudo-stable C-terminal helix extending from the folded core of the protein. We further describe the construction and characterization of a shorter construct that clearly demonstrates the unusual biophysical characteristics of
gpNu1
K100 result from this extended C-terminal helix. The biological
significance of these results is discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
where
(Eq. 1)
is the molar ellipticity
(degrees-cm2/dmol),
obs is the ellipticity
recorded by the instrument (millidegrees), MRW is the mean residue
weight (formula weight divided by the total number of residues in the
protein), b is the cell path length, and c is the
protein concentration in mg/ml (36). Protein secondary matrix-assisted
laser desorption time-of-flight mass spectra were obtained from the
University of Colorado Health Sciences Center Macromolecular Resource
Center. Automated DNA sequence analysis was performed by the University
of Colorado Cancer Center Macromolecular Resources Core facility. Both
strands of the duplex were examined to ensure the expected DNA
sequence. Prediction of protein secondary structures based upon primary
sequence data was performed by the method of Chou and Fasman (37),
using the DNASIS program (Macintosh version 2.0). Calculation of
protein secondary structures based upon the far-UV CD data was
performed using the SELCON program.
E68--
A truncated Nu1 gene
was amplified by PCR using pSF1 as a DNA template. This plasmid
contains the wild-type Nu1 gene cloned into a pBR322
background (38). Primers were designed such that EcoRI and
BamHI restriction sequences were present at the 5' and 3'
ends, respectively, of the PCR product. The primer sequences used to
amplify pNu1
E68 were as follows. Forward primer: 5'-CCT CTC CCT TTC
TCC GAA TTC ATG GAA GTC AAC AAA AAG
C-3'; reverse primer: 5-CTT CCT GGA TTC TTA
TTC TTC AAC CTC CCG GCG-3'. The EcoRI and
BamHI restriction sequences in the above primers are
indicated in italics, whereas the f-MET (forward primer) and stop (reverse primer) codons are shown in boldface.
Sequences complementary to the Nu1 gene are
underlined. The stop codon present in the reverse PCR primer
yields, upon amplification, a truncated Nu1 gene that
expresses only the first 68 amino acids of the protein. PCR
amplification, isolation of the PCR product, and construction of the
overexpression plasmid (pNu1
E68) was performed as described previously (1, 28, 35).
E68--
Four liters of
2X-YT media containing 50 µg/ml ampicillin, 25 mM
potassium phosphate, pH 7.5, and 5 mM glucose were
inoculated with a 40 ml of overnight culture of BL21(DE3)[pNu1
E68]
derived from an isolated colony. The cultures were maintained at
37 °C until an optical density 1.0 (A600 nm) was obtained, at which point
isopropyl-1-thio-
-D-galactopyranoside (1.2 mM) was added. The cells were maintained at 37 °C for an
additional 3 h, and then harvested by centrifugation. Unless
otherwise indicated, all subsequent steps were performed at 0-4 °C.
The cell pellet was resuspended in ice-cold buffer A (20 mM
Tris, pH 8.0, 2 mM EDTA, 7 mM
2-mercaptoethanol, and 10% glycerol) containing 100 mM
NaCl, and the cells were disrupted by sonification. Insoluble cellular
debris was removed by centrifugation (12,000 × g, 30 min), and solid ammonium sulfate was added to the clarified supernatant to 50% saturation. Insoluble protein was removed by centrifugation (12,000 × g, 30 min), and proteins were then
precipitated with the addition of ammonium sulfate to 90% saturation
followed by centrifugation. gpNu1
E68 was found in the 50-90%
ammonium sulfate-precipitated fractions.
E68 eluting at
300 mM NaCl. Column
fractions were examined by SDS-PAGE, and the appropriate fractions were
pooled, dialyzed against buffer A, and loaded onto an SP-Sepharose
column equilibrated with the same buffer. The column was developed with
a salt gradient with gpNu1
E68 eluting at
280 mM NaCl.
As before, column fractions were examined by SDS-PAGE, the appropriate
fractions were pooled, dialyzed against buffer A containing 20%
glycerol, and stored at
80 °C. As required, the proteins were
concentrated and/or buffer exchanged using an Ultrafree-15 centrifugal
filter device according to the manufacturer's instructions (Millipore).
1.2 mg/ml).
Samples were dialyzed against the appropriate buffer and then diluted
to the concentrations indicated in each experiment. Sample volumes were
100 µl with the inert oil FC-43 used to displace samples from the
base of the cells. Samples were allowed to equilibrate at 20,000, 30,000, and 40,000 rpm. Samples were judged to be at equilibrium by
successive subtraction of scans. The density of each buffer solution
was calculated based on the salt composition and equilibrium
temperature. The partial specific volume of gpNu1
E68 was calculated
by summing the partial specific volumes of the individual amino acids
(39). Data chosen for analysis had an absorbance between 0.1 and 1.5 optical density units. Each data point was an average of four scans
taken every 0.001 cm. Data were selected for analysis using the program
REEDIT (generously provided by Dr. David Yphantis). Individual and
simultaneous analyses of nine channels (three concentrations at three
speeds) were carried out to resolve assembly stoichiometry. Data were
analyzed using the appropriate functions by non-linear least-squares
parameter estimation (40) to determine the best-fit
model-dependent parameters that minimize the variance. The
program NONLIN was used (Ref. 41; kindly donated by Dr. David
Yphantis). Confidence intervals (67%) correspond to approximately one
standard deviation. Non-ideality was not considered, because there
was no evidence for non-ideal effects.
where Y(r) is the absorbance at radius
r,
(Eq. 2)
the baseline offset, and
the monomer absorbance
at reference radius ro.
is the reduced
molecular weight (
= M(1
)
2/RT], N is the
stoichiometry of the reaction, and KN is the
association constant of the reaction NM
MN.
where
(Eq. 3)
T is the ellipticity at temperature
T, and
N and
D represent the
ellipticity for the native and denatured protein, respectively.
Baseline corrections were not performed to demonstrate temperature-induced alterations in the pre-transition baseline slopes.
The unfolding curves were analyzed using a complex sigmoidal curve
function,
where (mN*T
(Eq. 4)
bN) and (mD*T
bD) describe the linear portion of the
pre-transition and post-transition baselines, respectively, at
temperature T, mT is the slope of
curve within the transition region, and Tm is
the melting temperature for the transition. All data sets were fit to
the above equations by non-linear regression methods using the IGOR
graphics/analysis package (WaveMetrics, Lake Oswego, OR).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K100, a
deletion construct of the small terminase subunit (1). The construct is
a dimer in the concentration range of 5 µM to 2 mM, with no evidence for dissociation or further
aggregation. Preliminary NMR experiments suggested that the construct
might be amenable to structural studies, and we therefore sought to
further characterize the physical properties of this construct.
K100--
Thermally induced
unfolding of gpNu1
K100 secondary and tertiary structural elements is
reversible, as indicated in Fig. 2, A and B,
respectively. Moreover, the loss of tertiary structure (near-UV CD
signal) is cooperative, consistent with a folded and stable construct
(Fig. 2C).2 Salt and protons
stabilize protein tertiary structure, as evidenced by the
significant increase in the Tm for the
transition (Table I). Despite the
observed cooperative loss of tertiary structure, thermally induced loss
of secondary structural elements (far-UV CD signal) appears essentially
non-cooperative (Fig. 2C). The steep pre-transition baseline
observed in these data make it difficult to accurately calculate the
Tm for this transition. Nevertheless, it is
clear that salt and protons similarly affect the
Tm for the unfolding transition, whether
monitored in the far-UV or near-UV region of the CD spectrum (Table I).
Interestingly, salt and pH strongly affect the pre-transition baselines
obtained in the far-UV CD melting curves, but not the near-UV CD
melting curves (Table II).
Salt and protons affect the thermal stability of gpNu1K100
Salt and protons affect the pre-transition baseline for thermally
induced gpNu1K100 secondary structure loss
K100--
Evaluation of the line
widths and chemical shifts in a 1H-15N
correlation spectrum of gpNu1
K100 suggested that
30 residues of
the construct were solvent exposed and highly
flexible.3 Primary sequence
analysis predicts strong
-helical character in the region spanning
residues
50 and
115 of gpNu1 (Fig.
1B). We postulated that this
putative helix might be partially disrupted in the gpNu1
K100
construct, leading to the unusual unfolding properties of the protein.
If this were the case, limited proteolysis of gpNu1
K100 would be
expected to degrade the exposed portion of the helix, yielding a fully
folded domain suitable for structural characterization. This was indeed
correct. Limited proteolysis of the gpNu1
K100 with a number of
proteases consistently yielded two predominant products (data not
shown). Analysis of these products by SDS-PAGE and matrix-assisted
laser desorption time-of-flight mass spectrometry yielded molecular
masses of
7.5 kDa and
10 kDa, respectively.
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Fig. 1.
A, model for terminase assembly at
cos. Details are given in the text. B,
domain organization and predicted secondary structure of gpNu1.
Functional domains are indicated in the upper box. The
helix-turn-helix motif (Lys5-Glu24)
identified by primary sequence analysis is also indicated. Secondary
structural elements were predicted by the method of Chou and Fasman
(37).
E68--
Proteolysis studies, NMR
spectral analysis, and evaluation of secondary structure predictions
for gpNu1
K100 suggested that the minimal folded DNA binding domain
of gpNu1 is located within the N-terminal
70 amino acids of the
protein. Based on these data, gpNu1
E68 was constructed. Expression
and purification of this construct as described under "Experimental
Procedures" yielded 10 mg of homogenous protein per liter of cell growth.
E68 is typical of a purified
protein that is essentially devoid of contaminating nucleotide (A280:A260 = 1.95) (42).
An extinction coefficient of
280 = 13.9 mM
1·cm
1 was calculated for
the protein using the method of Gill and von Hippel (Table
III) (43, 44). The progressive decrease
in
280 going from full-length gpNu1, to gpNu1
K100, to
gpNu1
E68 is consistent with deletion of one Tyr and four Phe in
gpNu1
K100, and an additional Tyr in gpNu1
E68. The fluorescence
spectrum of gpNu1
E68 exhibits a maximum of 335 nm using an
excitation wavelength of 280 nm (Table III). This maximum blue shifts
to 350 nm and increases in intensity (1.6-fold) in the presence of 6 M guanidinium hydrochloride, consistent with denaturation
of a folded protein. Identical fluorescence changes are observed with
full-length gpNu1 and the gpNu1
K100 construct (Table III),
suggesting that the folded core of all the proteins is similar.
Importantly, gpNu1
E68 binds cos-containing DNA with an
affinity essentially identical to that of
gpNu1
K100.4
Spectral properties of the gpNu1 constructs
-Helical content was obtained by
deconvolution of the far-UV CD spectra as described under
"Experimental Procedures."
E68 demonstrates that the protein
possesses secondary structural elements (Fig. 3A).
Deconvolution analysis of the spectrum is consistent with a protein
containing 30%
-helical structure as well as 22% of the residues
being in a
-sheet conformation. This represents a significant loss
of
-helical character compared with that observed in the
gpNu1
K100 construct (Table III). Strong signals are also observed in
the near-UV CD spectrum of the construct (Fig. 3B),
demonstrating that the protein also possesses significant tertiary
structure. The near-UV CD spectrum of gpNu1
E68 is quite similar to
that of gpNu1
K100, with the exception of
decreased signal intensity at 276 and 282 nm (compare Figs. 2B and
3B). These bands likely represent the 1Lb transitions of Tyr-84, which
has been deleted in the gpNu1
E68 construct.
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Fig. 2.
A, far-UV CD spectra of gpNu1 K100
before (
) and after (x) heating to 85 °C for 15 min.
B, near-UV CD spectra of gpNu1
K100 before (
) and after
(x) heating to 85 °C for 15 min. The spectra presented in
A and B were recorded at 4 °C. C,
thermally induced unfolding of gpNu1
K100. Unfolding was monitored by
far-UV (
) and near-UV (
) CD signals as described under
"Experimental Procedures."
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Fig. 3.
A, far-UV CD spectra of gpNu1 E68
before (
) and after (x) heating to 85 °C for 15 min.
B, near-UV CD spectra of gpNu1
E68 before (
) and after
(x) heating to 85 °C for 15 min. The spectra presented in
A and B were recorded at 4 °C. C,
thermally induced unfolding of gpNu1
E68. Unfolding was monitored by
far-UV (
) and near-UV (
) CD signals as described under
"Experimental Procedures."
E68 was determined by sedimentation equilibrium
methods. Least-squares analysis of a representative data set is
presented in Fig. 4 and Table IV. The
data consist of three different loading
concentrations at three different rotor speeds as described under
"Experimental Procedures." Each individual data set was fit to a
single-species monomer model to resolve an average molecular mass. The
apparent molecular mass is constant and twice that of the calculated
monomer molecular mass of 7,803 Da, indicating that the
construct is largely dimeric at all concentrations examined.
Furthermore, there is no evidence of a
concentration-dependent increase in molecular mass,
indicating that gpNu1
E68 dimers are not in dynamic equilibrium with
either monomers or higher order assemblies. A more rigorous analysis by
simultaneous fitting of all nine data sets to a single-species model
resolved an apparent molecular mass of 17,665 ± 749 Da, 2.26-fold
greater than the calculated monomer molecular mass. The quality of the
simultaneous analysis can be seen in Fig. 4, with all data being well
described by the model. The square root of the variance for the
simultaneous analysis (0.010 optical density units) is also comparable
to that of any individual fit, indicative of no systematic deviations. Fitting of the data to more complex assembly schemes either resulted in
no improvement of the fit, or failure to converge. Taken together, the
data demonstrate that gpNu1
E68 is exclusively dimeric over the
concentration range of 15 to 150 µM. Identical results
were obtained in the absence or presence of 150 mM NaCl,
and at temperatures between 4 °C and 37 °C.
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Fig. 4.
Sedimentation equilibrium analysis of
gpNu1 E68. Data sets represent initial
loading concentration of 150 µM (left), 45 µM (center), and 15 µM
(right) in 10 mM sodium phosphate buffer, pH
7.0, containing 150 mM NaCl. The symbols
represent absorbance of gpNu1
E68 equilibrated at a rotor speed of
20,000 (
), 30,000 (
), and 40,000 (
) rpm, respectively. For
clarity, only every fifth data point is shown. The data were acquired
at 4 °C and analyzed as described under "Experimental
Procedures." The solid lines represent the best-fit to a
single species model obtained from simultaneous analysis of all nine
data sets.
Sedimentation equilibrium analysis of gpNu1E68
E68--
Thermally induced
loss of gpNu1
E68 secondary and tertiary structure is fully
reversible (Figs. 3, A and B). The loss of
tertiary structure is cooperative (Fig. 3C) and possesses a
Tm similar to that observed for the gpNu1
K100
construct (compare Tables I and V).
Moreover, salt and protons similarly affect the thermal stability of
both constructs. Thermally induced loss of gpNu1
E68 secondary
structures is also cooperative, and the pre-transition baseline is
comparatively insensitive to temperature (compare Figs. 2C
and 3C). Moreover, the pre-transition baseline for
gpNu1
E68 unfolding is unaffected by salt or pH (not shown). A
significant increase in Tm is observed with
increasing gpNu1
E68 concentration (Table
VI). Essentially identical results are
observed with gpNu1
K100.
Salt and protons affect the thermal stability of gpNu1E68
Protein concentration affects the thermal stability of the gpNu1
constructs
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K100, a deletion mutant of
gpNu1 that comprises the N-terminal 100 amino acids of the protein.
This construct is fully soluble at concentrations up to 15 mg/ml (1),
which allows biophysical and structural studies that are not
possible with the full-length protein. Importantly, gpNu1
K100
contains the putative helix-turn-helix DNA binding motif found in
full-length gpNu1 (see Fig. 1B) (45). We therefore initiated
NMR structural studies on this construct to understand the structural
basis for the stability of gpNu1·DNA complexes.
K100 reveals an unusual situation where
secondary structure is lost in a non-cooperative manner, whereas global
folding of the protein (tertiary structure) is lost cooperatively.
Based on a variety of data, we reasoned that gpNu1
K100 is composed
of a stable and folded N-terminal domain that possesses a C-terminal
helix extending from the folded core of the protein (Fig.
5). Specifically, the C-terminal 30 amino acids of gpNu1
K100 adopt a pseudo-stable helix that "unravels" in response to elevated temperatures, much like an isolated helical peptide. This non-cooperative loss of secondary structure is reflected in the steep pre-transition baseline observed in the far-UV CD melting
curves. Ultimately, the folded N-terminal region of the protein
denatures, yielding the cooperative unfolding curves observed in the
near-UV CD unfolding transition.
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Fig. 5.
Model for the structural organization of the
terminase gpNu1 subunit. HTH indicates the putative
helix-turn-helix DNA binding motif.
Limited proteolysis experiments indeed demonstrate the presence of a
protease-sensitive C terminus, and the shorter gpNu1E68 mutant was
constructed. Fluorescence, CD, and NMR data confirm that this construct
is folded in solution. gpNu1
E68 retains a dimeric structure under
all experimental conditions examined, indicating that the
self-association domain of full-length gpNu1 (Lys100-Pro141) is distinct from the
dimerization region of the protein. Our data do not allow the
calculation of an equilibrium constant for protein dimerization but
suggest an upper limit in the nanomolar range. Of interest is the
observation that thermally induced unfolding of both constructs is
concentration-dependent, suggesting that unfolding and
dimerization are, at least in part, coupled.
Thermally induced loss of gpNu1E68 secondary structure is
cooperative, unlike that observed for gpNu1
K100. Moreover,
gpNu1
E68 unfolding monitored in the near-UV CD mirrors that obtained
in the far-UV CD, suggesting that loss of secondary structure is concomitant with global unfolding of the protein. This is consistent with the suggestion that the strongly sloping baselines in gpNu1
K100 unfolding curves result from loss of secondary structure as the pseudo-stable helix unwinds. Deletion of this region results in cooperative secondary structure loss as the entire protein unfolds.
Our data suggest that the tertiary fold of the N-terminal 68 residues
in both gpNu1E68 and gpNu1
K100 is essentially identical. First,
guanidinium hydrochloride-induced denaturation yields identical fluorescence changes in both constructs. Second, the near-UV CD spectra
confirm the loss of tyrosine 85 from gpNu1
E68 but otherwise indicate
similar folded structures for the two constructs. Finally, thermally
induced unfolding of tertiary structure yields comparable Tm values for both constructs, and the salt and
pH effects are virtually identical. These data suggest that deletion of
residues 69 to 100 from gpNu1
K100 does not appreciably affect the
folding or stability of the N-terminal 68 residues of the protein.
Consistently, the affinity of both constructs for
cos-containing DNA is essentially identical.
The data are consistent with the model presented in Fig. 5 that
describes gpNu1E68 as a folded, stable, and functional DNA binding
domain of gpNu1. The model further suggests that the C-terminal 32 amino acids of gpNu1
K100 adopt a pseudo-stable helix that extends
from the folded core of the protein. This is consistent with primary
sequence analysis that predicts strong
-helical character extending
from residue
50 in the DNA binding domain to residue
115 within
the self-association domain of full-length gpNu1. Truncation of the
helix at Glu100 yields a disrupted helix that is marginally stable.
Of what functional significance is this helical region of gpNu1? The
proposed structural organization of gpNu1 finds similarity to that
observed in the subunit of E. coli RNA polymerase (46). This protein consists of two independently folded domains connected by
a flexible sequence that possess
-helical character. The N-terminal domain (NTD) forms a homodimer that is necessary and sufficient for
core enzyme assembly and site-specific DNA binding. The C-terminal domain (CTD) is responsible for trans-activation by a number
of transcription factors. The intervening flexible helix allows the CTDs to act as independent motional units capable of interacting with a variety of protein signals, whereas the NTD remains
site-specifically bound at the promoter. A similar structural
organization is observed in the integrase enzyme from HIV. The dimeric
core domains are connected to the C-terminal DNA binding domains by a
26 residue
-helix (47). In this case, the interdomain helix likely
plays a functional role that permits dynamic interaction of the CTDs during the integration of viral DNA.
We suggest a similar role for an intervening helix in gpNu1. The
N-terminal domain forms a homodimer that binds specifically to
cos-containing DNA. Cooperative DNA binding is driven by
self-association interactions mediated by the hydrophobic domain of the
protein. Additionally, the C-terminal gpA-interactive domain of gpNu1
must promote gpA assembly at cosN. A helical linker between
these domains would allow sufficient flexibility such that each of
these contacts may be formed appropriately, simultaneously assembling
gpNu1 and gpA at cosB and cosN, respectively.
Structural, kinetic, and biophysical studies currently underway in our
laboratory are directed toward a mechanistic description of the role of
this helix in the functioning of the protein, and virus assembly.
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ACKNOWLEDGEMENT |
---|
We are indebted to Dr. Mark Manning for use of the CD spectrometer used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB-9728550.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Colorado Health Sciences Center, School of Pharmacy, 4200 East Ninth Ave., Campus Box C238, Denver, CO 80262. Tel.: 303-315-8561; Fax: 303-315-6281; E-mail: carlos.catalano@uchsc.edu.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100517200
3 T. de Beer and C. E. Catalano, unpublished.
4 T. de Beer and C.E. Catalano, manuscript in preparation.
2
gpNu1K100 possesses two tryptophan
(Trp22 and Trp49) and three tyrosine (Tyr41,
Tyr50, and Tyr84) residues. The thermal unfolding data
provide no indication for multiple unfolding transitions, which would
indicate local unfolding in the vicinity of these residues. We thus
interpret the loss of the near-UV CD signal as reflecting global
unfolding of the protein. It is feasible, however, that the melting
curves reflect regional versus global unfolding of the protein.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
kb, kilobase(s);
cos, cohesive end site, the junction between individual
genomes in immature concatemeric DNA;
gpA, the large subunit of
phage
terminase;
gpNu1, the small subunit of phage
terminase;
gpNu1
E68, a gpNu1 construct truncated at Glu68;
gpNu1
K100, a gpNu1 construct truncated at Lys100;
gpNu1
P141, a gpNu1 construct truncated at Pro141;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
NTD, N-terminal domain;
CTD, C-terminal domain;
CD, circular
dichroism.
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