Domain organization, folding and stability of bacteriophage T4
fibritin, a segmented coiled-coil protein
Sergei P. Boudko
1,2
, Yuri Y. Londer
1
, Andrei V. Letarov
1
, Natalia V. Sernova
1
, Juergen Engel
2
and Vadim V. Mesyanzhinov
1
1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia;
2
Biozentrum der Universitaet Basel, Switzerland
Fibritin is a segmented coiled-coil homotrimer of the
486-residue product of phage T4 gene wac.Thisprotein
attaches to a phage particle by the N-terminal region and
forms fibrous whiskers of 530 A
˚
, which perform a chaperone
function during virus assembly. The short C-terminal region
has a b-ann ulus-like structure. We engineered a set of fibritin
deletion mutants sequentially truncated from the N-termini,
and the mutants were s tudied by differential scanning
calorimetry (DSC) and CD measurements. The analysis
of DSC curves indicates that full-length fibritin exhibits
three thermal-heat-absorption peaks centred at 321 K

properly after heat denaturation. In contrast, fibritin XN,
which is deleted for the C-terminal domain, forms aggregates
inside the cell. The XN protein can be partially refolded by
dilution from urea and does not refold after heat denatur-
ation. These results confirm that the C-terminal domain is
essential for correct fibritin assembly both in vivo and in vitro
and a cts as a foldon.
Keywords: bacteriophage; foldon; microcalorimetry; protein
engineering; segmente d coiled coil.
Fibritin, a structural protein of bacteriophage T4 encoded
by gene wac (named for whisker’s antigen con trol), belongs
to a specific class of accessory proteins that act in the virus
assembly process. Six fibritin molecules form the collar/
whisker complex that consists of a ring embracing the phage
neck with thin filaments (whiskers) protruding from the
collar [1]. T his complex is a sensing device that controls th e
retraction of the long tail fibers in adverse environments and
thus prevents undesirable infection [2]. The whiskers act also
as a chaperone and help the proximal and distal parts of the
long tail fibers to join correctly by increasing the effective
target sizes and thereby increasing the rates of otherwise
slow diffusion–limited bimolecular interactions [3].
The structure of fibritin was predicted from sequence and
biochemical analyses to be mainly a parallel segmented
triple-helical coiled-coil [4,5]. Fibritin is a homotrimer of 486
residues per monomer and consists of three functional parts.
Its predominant central region has 1 3 consecutive a helical
coiled-coil segments linked by loops. The protein is attached
to a phage particle by the N-terminal part that does not
have heptad periodicity [6], and the short C-termini is

E-mail: [email protected]
Abbreviations: DSC, differential scanning calorimetry;
IPTG, isopropyl thio-b-
D
-galactoside.
(Received 20 July 2001, revised 6 December 2 001, accepted
11 December 2001)
Eur. J. Biochem. 269, 833–841 (2002) Ó FEBS 2002
suggested by the structural properties o f the coiled-coil
segments [8]. Trimerization is likely to begin with the
formation of the C-terminal domain that acts as a folding
nucleus domain (foldon) and subsequently initiates the
assembly of the coiled c oil [8,10]. The interplay between the
stabilizing effect of the C-terminal domain and the labile
coiled-coil domain may be essential for the fibritin function
and for the correct functioning of many other a helix fibrous
proteins as well.
In the present work, we o btained a set of fibritin mutants
sequentially truncated from the N-termini. We engineered
also mutant S1 that have deleted for one loop of 12 residues
in fibritin E. To characterize the thermodynamic properties,
stability, and domain organizations, we analysed these
fibritin mutants by d ifferential scanning calorimetry (DSC)
and CD measurements. The analysis of DSC curves
indicates that full-length fibritin has three thermal heat-
absorption transitions that were reasonably assigned to the
N-terminal, segmented coiled-coil, and C-terminal func-
tional domains, respectively.
Full-length fibritin and all the mutants truncated from the
N-termini refold p roperly a fter heat denaturation. We

Glu, Asn404-Glu415) was c onstructed on the basis of
fibritin E. We have used an overlap ping PCR method to
delete the DNA piece en coding this loop [13]. Sequencing
was carried out by the d ideoxy chain termination method
using a DNA sequencing kit/BigDye terminator cycle
sequencing ready reaction (Applied Biosystems) a nd an
automated DNA sequencer.
Expression and purification of fibritin mutants
The cell culture of the E. coli BL21 (DE3) strain carrying
the respective vector was grown at 37 °C in 500 mL of
2 · tryptone/yeast medium [14] until the density reached a
D
600
value of 0 .6. Protein expression was induced by 1 m
M
IPTG with subsequent incubation for 3 h at 37 °Cwith
vigorous aeration. We used a modification of the previously
Fig. 1. Schematic presentations and amino-acid sequence of fibritin.
(a) Schematic presen tation of th e fibritin mutants used in this work:
full-length fibritin ( wac), XN, B1, SM1, SM4, E, S1, M, and F. For
each mutant, the range of amino-acid sequence that it comprises of the
full-length fibritin sequence is given. The N-terminal domain is a broad
box; coiled-coil regions are narrow boxes; the loops, separating coiled-
coil segments, are hexamers; the C-terminal domain (foldon) is a
sphere. (b) Amino-acid sequence of full-length fibritin and heptad
scheme of the fibritin coiled coil part. The hydrophobic residues in the
a and d positions are shown in bold. The coiled-coil segments are
indicated by roman (I–XIII), and the loops are marked [L1–L11]. The
bacteriophage T4 gene wac nucleotide sequence is deposited in the
EMBL Ge ne Data Ba nk: accession number X12888. Atomic c oordi-

ing recombinant proteins, were dialysed against Tris/EDTA
buffer and stored at 4 °C. The E, S1 and F proteins were
additionally applied to a 15-mL DEAE–Sephacryl column
and eluted w ith a linear gradient o f NaCl. Fractions
containing proteins were dialysed against Tris/EDTA buffer
andstoredat4°C.
The protein purity was judged by denaturing SDS/PAGE
using two systems: for proteins with M
r
larger than 12 kDa
we used the Lae mmli system [15]; f or smaller ones we
applied the S chaegger and Jagow system [ 16]. Protein
concentration was determined by measuring the absorbency
at 280 nm in 6
M
GdnHCl, and the extinction coefficient
was c alculated a s described in [17]. For the DSC procedure
the proteins were dialysed against NaCl/P
i
[10 m
M
Na
phosphate (pH 8 .0), 150 m
M
NaCl or 10 m
M
Na phosphate
(pH 8.0)], centrifuged at 10 000 g for 30 min, and degassed
for 5 min.
Purification and refolding of the XN fibritin

TM
) of 0.5 mL volume at
a heating rate of 1 KÆmin
)1
. Baseline subtraction, calcu-
lation of DH
cal
for different peaks and determination of
absolute heat capacity were performed using the MicroCal
ORIGIN
5.0 program. To determine absolute heat capacity
of proteins, we used the following parameters in the
equation:
DC
p
¼ g
0
qðtÞV
0
ð1 þ 0:00002tÞ C
Abs
p
ðtÞÀvð1 þ atÞC
W
p
ðtÞ
hi
where DC
p
is the sample-buffer baseline minus the buffer-

)1
) (stored in Origin). The thermal
coefficient of cubic expansion of tantalum is 0.00002.
The values of the van’t Hoff enthalpy of the process for
the peaks representing the melting of coiled coil region were
calculated as for a first ord er reaction [19]:
D
1
0
H
vh
¼
4RT
2
max
ðhDC
p
i
max
À D
1
0
C
p
=2Þ
D
1
0
H
cal

equipped with a thermostatic quartz cell having a 1-mm
path length. CD data were analysed using the
CONTIN
program [20].
RESULTS
Engineering and properties of fibritin deletion mutants
To investigate the stability and thermodynamic properties
of T4 fibritin, a set of recombinant truncated mutants was
designed and analysed. All these molecules contained an
intact C-terminal part and had different numbers of coiled-
coil segments and separating segments loops (Fig. 1 and
Table 1). Fibritin S1, based on fibritin E with 120 resides per
chain, had a deleted loop L11 of 12 residues, and fibritin XN
hadnoC-terminalregionof30residues.
To enhance the prote in stability, five mutations were
introduced into the 74 residues of fibritin M that forms
the last coiled-coil segment (5,5 heptad repeats) and the
complete C-terminal domain [9]. Particularly, the Ser421
residue was substituted for Lys to test the possible
formation of interchain salt bridge with Glu426. The
substitutions Asn428 to Asp and Thr433 to Arg were
designed to create a similar interchain salt bridge b etween
these two residues. Residue 425, an Asp in a d position,
was replaced by an Ile, which is generally a favourable
residue in this position for a trimeric coile d coil [21].
Ó FEBS 2002 Thermodynamics of segmented coiled coil protein (Eur. J. Biochem. 269) 835
The crystal structure of two fibritin truncated mutants,
E and M, that have 120 and 74 residues per monomer,
respectively, have been determined to a tomic resolution [ 8].
X-ray crystallography confirmed that both m utants are

decreased with decre asing size of the mutants. The mean
residue elliptic ity at 220 nm was )32 800 degÆcm
2
Ædmol
)1
for full-length fibritin and )25 800 and )21 900 degÆcm
2
Æ
dmol
)1
for fibritin B1 and fibritin SM4, respectively.
Interestingly, fibritin M e xhibited more a helicity than
fibritin E, probably due to the absence of insertion loops.
The CD spectrum of fibritin F represented mostly the
secondary structure of t he C-terminal domain, which is in a
good agreement with published data [22].
Assignment of the fibritin thermal transitions
to functional domains
The full-length fibritin, and the N-te rminally truncated B1,
SM1, SM4, E, M, and F mutants were a nalyzed by DSC.
The DSC data were also collected for fibritin XN that had
no C-terminal domain. Our goal was to answer a question
about how many thermodynamically independent domains
fibritin has, and to assign the thermal transitions to
individual functional regions. M easurements were per-
formed in 10 m
M
Na pho sphate buffer, pH 8.0 with
0.15
M

residues
DH
cal
of all transitions
(total) (JÆmol
)1
)
DH
cal
coiled-coil
transition (JÆmol
)1
)
DH
cal
folding
nucleus (JÆmol
)1
)
DH
vh
/DH
cal
coiled-coil
transition
Wac 486 )9280 )7600 – 0.13
B1 281 )3610 )3080 )530 0.39
SM1 258 )3170 )2640 )530 0.42
SM4 182 )1660 )1170 )490 0.88
E 120 – )687 – 0.91

larger than the other two transitions at 321 K and 345 K,
and it definitely can be assigned to the coiled-coil part. The
occurrence of only a single transition strongly supports
co-operative heat-induced unfolding of all coiled coil
segments. Unfolding of the coiled coil of fibritin XN gave
two heat a bsorption peaks centred at 330 K and at 336 K
(see below). The appearance of the 330 K transition can be
explained b y the structure destabilization at the C-terminus
due to the elimination of 30 l ast residues.
Besides the 345 K peak, fibritin B1, which consisted
about half of a full-length molecule (Fig. 1), as well as
shorter SM1 and SM4 mutants all had another heat
absorption peak with a midpoint at 330 K. (Fig. 3A).
However, for fibritin E this peak was centred at 320 K, and
the smallest fibritin M and F showed no separation of
melting between the C-terminal domain and the coiled-coil
region (Fig. 3A). Significant stabilization of fibritin M, in
comparison with a wild-type fibritin, can be explained
mainly by two residues substitutions. As confirmed by
X-ray crystallography [9]
,
the mutation Ser421 to Lys
created a new salt bridge between residues Lys421 and
Glu426. These residues occupy the g and e heptad’s
positions in different chains within fibritin M trimer. It is
known that interchain salt bridges have a stabilizing effect
on the coiled coil [23]. Anoth er mutation, Asn425 to Ile,
Fig. 4. The calorimetric enthalpy plots for the full-length fibritin (wac), B 1, SM1, SM4 , and F proteins in 0.01
M
Na phosphate buffer (pH 8.0) and

also seems to increase the stability o f fibritin M.
The DH
cal
values of the 336 K peak of full-length fibritin,
andofthe330KpeaksoftheB1,SM1,SM4,andE
truncated molecules were proportional t o their size (Fig. 5).
The m ean enthalpy, calculated from the slope of the graph,
was DH
res
¼ 21 kJÆmol residue
)1
. The singularity a nd pro-
portionality of that transition are consistent with the
thermal unfolding of a uniform do main. By varying the
ionic strength of the sample buffer, no discrete melting of
subdomains was found for the short coiled-coil segments
(data not shown).
The melting temperature of the coiled-coil region of the
B1,SM1,SM4(T
m
¼ 33 0 K), and E (T
m
¼ 320 K)
mutants was lower than that for the respective part of a
wild-type fibritin (T
m
¼ 336 K). This was an indication that
the deletion of the N-terminal sequence of fibritin had a
destabilizing influence. The ratio of DH
VH and

¼ 327–328 K) (data not shown). At the present, by
varying pH and ionic strength conditions, we are trying to
detect subdomain transitions of the coiled-coil region.
Stability of the S1 fibritin
Three coiled coil segments of fibritin E are separated by
two loops: r esidues Gly386–Gly391 form the first one
(L10) and the second one (L11) contains the residues
Asn404–Gly417 [9] (Fig. 1). To clarify the role of the loop
regions in protein stability, we designed fibritin S1 lacking
the Asn-Gly-Thr-Asn-Pro-Asn-Gly-Se r-Thr-Val-Glu-Glu
sequence of loop L11 [13]. The two last L11 loop residues,
ArgandGly,werepreservedinS1tomadethecoiledcoil
continuous (Fig. 1B).
The calorimetric transitions for the coiled-coil regions of
the E and S 1 mutants differed by 10 K ( Fig. 3B). The
coiled-coil part, which lacked the loop sequence, melted at
330 K while fibritin E had a transition at 320 K. The
enthalpy of this transition was DH
cal
¼ 656 kJÆ(mol
trimer)
)1
for fibritin S1 and 687 kJÆ(mol trimer)
)1
for
fibritin E. Most probably, the stability of S1 increased due
to the f ormation of uniform coiled coil containing two
segments, XI and XII. Also, e limination of loop 11 might
have helped to form of additional salt bridge between
residues Glu435 and Lys440, at the g and e positions,

(Fig. 6 A). In contrast, all fibritin mutants containing the
C-terminal domain exhibited reversible refolding under t he
Fig. 5. Far CD spectra for the SM4, and F proteins and folding nucleus
alone in a solution of 0.01
M
Na phosphate buffer (pH 8.0) and 0.15
M
NaCl. (a) Spectra of the SM4 fibritin (182 residues per monomer)
were registered at 298, 335, and 358 K. The protein has the native
conformation at 298 K, and is completely unfolded at 358 K. The
335 K spectrum is the spectrum of the partially unfolded state in
which the coiled-coil part is disordered and the folding nucleus
domain still has its sec ondary structure. T his may b e seen at 229 nm:
the 335 K spectrum has a more positive h-value than the 358 K
spectrum. The difference of the signals for these two spectra assigned
only for the folding n ucleus (30 residues) is presented in (b) in
comparison with the isolated the C-terminal part spectra [22]. The
C-termini peak, centred at 229 nm, can easily be detected also for
fragment F th at h as o nly 5 8 residues p er mo no mer ( a).
838 S. P. Boudko et al. (Eur. J. Biochem. 269) Ó FEBS 2002
same conditions. As an example, Fig. 6B shows the results
of heat denaturation of fibritin B1. After heating to 336 K,
the transition curves for second and third rounds differed
from the first one by only a few percent. The d ifferences
were even smaller f or shorter fibritin fragments. Significant
flattening of th e peaks corresponding to the coiled-coil
region was observed only a fter heating to 369 K (see
Fig. 6B, f or fibritin B1). Prolonged heating led to a further
decrease of the extent o f refolding. I ndependent of temper-
ature and time of heat exposure, refolding of the C-terminal

domain of fibritin B1 do not unfold in an all-or-none
manner. ÔNon all-or-none transitionÕ means that we do have
intermediates, but in the case of fibritin and other fibrous
proteins these intermediates do not have fixed structures
because these proteins have a zipper-like mechanism of
folding-unfolding [27]. Nevertheless, the sharpness of the
transition and t he failure to detect a splitting of the
transition profile i nto individu al subpeaks suggests that
loop regions, connecting B1 coiled-coil segments, serve as
co-operative linkers between the segments. According to
equilibrium criteria, the unfolding and reversible refolding
of the nine segments therefore occurs in a s ingle step.
The s ingularity of the coiled-coil transition, midpoint
temperature and peak sharpness are maintained also for the
SM1 and S M4 fibritins in which the nu mber of coiled-coil
segments is reduced to eight and five, respectively. The all-
or-none approximation is better f ulfilled for these p roteins,
which is expe cted for th eir smaller size and more limited
contacts. Interestingly, the enthalpy of the transition f or the
E, SM4, SM1 and B1 fibritins increases linearly with an
increasing number of amino-acid r esidues in the coiled-coil
region. In contrast to the independent melting of the coiled-
coil segments of different stability, this is additional evidence
for the co-operative transition of the e ntire coiled-coil
region. The ratio of the van’t Hoff enthalpy to calorimetric
enthalpy for fibritin E is 0.91, is very close to 1 for the all-
or-none approximation. This finding, which is in accordance
with the crystallographic observation [8] that two coiled-coil
segments of fibritin E is a repetitive structured domain with
loop regions as a part of the structure. The e nthalpy change

fibritin assembly in vivo and in vitro and act as a f oldon.
Foldon is a protein unit that forms on the initial steps of
folding [31,32] which frequently perform a specific, distinct
function that remains intact even after isolated or trans-
ferred into other proteins [22,33–35]. The stabilizing and
assembly of the trimeric T4 fibritin foldon has been
demonstrated recently by protein engineering for several
chimera proteins [22,36,37].
ACKNOWLEDGEMENTS
We thank Dr Kyle Tanner for critical reading of the manuscript,
and Dr Sergei Yu. Venyaminov for providing the CONTIN
program. This work was supported in part by HHMI (grants
75195–52080, and 55000324), Russian Foundation for Basic
Research (grant 99-04-4843 0), and by the ÔUniversities of Ru ssiaÕ
grant to V. V. M, and by Swiss National Science Foundation (grant
31-49281.96) to J. E.
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