Báo cáo Y học: Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein docx

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 (DH ¼ 1390 kJÆmol trimer )1 ), at 336 K (DH ¼ 7600 kJÆmol trimer )1 ), and at 345 K (DH ¼ 515 kJÆmo l trimer )1 ). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy DH res ¼ 21 kJÆmol residue )1 .TheratioofDH VH /DH cal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.8 8, 0.42, 0.39, and 0.13, respectively. This gives an indication of the d ecrease of the Ôall-or-noneÕ character of the transition with increasing protein s ize. The deletion o f the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the m ut ants t runcated f rom the N- termini ref old 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 essential for in vivo protein folding and trimerization [5]. Functional activities of fibritin can be related to the exposure of hydrophobic patches in the c oiled-coil [7]. The full-length fibritin of 530 A ˚ could not be crystallized, probably because of its inherent flexibility. However, a set of smaller fibritin mutants was engineered and expressed in the soluble trimeric forms in an Escherichia coli system [5,8,9]. The structures of the E and M fibritins, which are truncated for the last 120 and 75, respectively, C-terminal residues per monomer were solved to atomic resolution by X-ray crystallography [8,9]. Three identical subunits form a trimeric p arallel coiled-coil domain and a small a structural C-terminal domain. The coiled-coil part of fibritin E is divided into three segments separated by short sequences called insertion loops. The C-terminal domain, which consists of 30 residues from e ach monomer, contains a b-annulus-like structure with a hydrophobic interior. Residues within the C-terminal domain make extensive hydrophobic and some polar inter–subunit interactions [8]. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown by mutational studies ([5] and S. P. Boudko, unpublished results). Tight interactions between C-terminal residues of adjacent subunits counteract the latent instability that is Correspondence to V. V. Mesyanzhinov, Howard Hughes Medical Institute, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Street 16/10, 117997 Moscow, Russia. Fax: + 7 095 336 6022, Tel.: + 7 095 335 5 588, E-mail: vvm@ibch.ru 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 designed also the XN mutant, a full-length fibritin that has no C-terminal domain (Fig. 1) that forms aggregates inside the cell. The XN protein can be partially refolded by fast dilution from urea and does not refold after heat denatur- ation. The XN protein can be refolded by fast dilution from urea and does not refold after heat d enaturation. MATERIALS AND METHODS E. coli strains and plasmids The Top10 E. coli strain (Invitrogen, USA) was used for the selection of recombinant clones and plasmid DNA purifi- cation. Protein expression was performed in the BL21 (DE3) strain (Promega, USA) containing the T7 RNA polymerase gene under lac UV5 control in the E. coli chromosome. DNA fragments encoding truncated fibritin mutants were cloned in the pET19b (+) and pET23d (+) expression vectors containing the ribosome-binding site for effective translation (Novagen, U SA), that allow transcrip- tion from the T7 RNA polymerase promoter. Design of fibritin mutants We used previously designed expression vectors for a full- length fibritin [8], fibritin XN [10,11], E, M [8,9], F (V. V. Mesyanzhinov, unpublished results), and the S1 fibritin [12,13]. To create the B1, SM1, SM4 mutants, we amplified the DNA fragments of interest by PCR and introduce the NcoI and BamHI restriction sites for subsequent cloning into plasmid vectors. Cloning was performed using the common t echniques described in [14]. The S1 mutant that lacks 12 residues of the L11 loop (residues Asn-Gly-Thr-Asn-Pro-Asn-Gly-Ser-Thr-Val-Glu- 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- nates of fibritin E and fibritin M, deposited in PDB, are 1AA0 and 1AVY, respectively. 834 S. P. Boudko et al. (Eur. J. Biochem. 269) Ó FEBS 2002 described method for purification of fibritin mutants [5]. The pellet from 500 mL of the E. coli culture was resuspended in 10 mL of Tris/EDTA buffer (50 m M Tris/HCl, pH 8.0, 1 m M EDTA) and sonicated with cooling. The cell debris was removed by centrifugation at 25 000 g for 20 min. T o precipitate nucleic acids, 1 mL of 30% (w/v) streptomycin sulfate (Sigma, USA) solution in Tris/EDTA buffer was added; the concentrated protein solution was kept on ice for 15 min. After centrifugation, ammonium sulfate was added to the supernatant to a final concentration of 20–50% saturation, depending on the particular mutant, and the mixture was incubated overnight at 4 °C. Protein precipitate was collected by low-speed centrifugation, and resuspended in 3–10 mL o f Tris/EDTA buffer. Nucleic acid and protein precipitation procedures were skipped f or protein S1. After ammonium sulfate precipitation, the protein solution was applied to a 10-mL hydroxyapatite column (Bio-Rad; DNA grade) equilibrated with 10 m M Na phosphate (pH 8.0) and was hed with 10 m M Na phosphate. The flow-through fractions, contain- 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 The pellet from 500 mL of the E. coli cells expressing fibritin XN was suspended in 10 mL of Tris/EDTA buffer ( 50 m M Tris/HCl (pH 8.0), 1 m M EDTA) and sonicated under cooling. The cell extract was centrifuged at 3500 g for 30 min and supernatant was r emoved. The pellet was resuspended in 0.5 mL of 8 M urea for 10 min and the suspension was centrifuged at 10 000 g for 30 min to remove insoluble particles. The supernatant was mixed with 50 mL of the refolding buffer (50 m M Tris/HCl, pH 8 .0, 2m M EDTA, 2 m M phenylmethanesulfonyl fluoride), incubated at 4 °C for 3–4 days and then concentrated to 2 m L. The protein solution was further purified on the hydroxyapatite column as described a bove. The yield of the soluble protein was % 15% o f initial concentration i ndicat- ing weak refolding. DSC Calorimetric measurements were performed using a VP-DSC Microcalorimeter (Microcal Inc.) equipped with a cell (covered with Tantaloy 61 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- buffer baseline, g 0 is the c oncentration o f protein (g ÆmL )1 ), q(t) is the relative density of water (stored in the ORIGIN program [18]), V 0 is the nominal volume (0.5194 mL) of the sample cell, t is temperature in °C, C Abs p (t)istheabsolute heat capacity (calÆdeg )1 Æg )1 ) o f the protein i n solution, v is the partial specific volume of the protein (0.717 mLÆmg )1 ), a is the coefficient of thermal expansion of the protein (0.0007 1/ a °C), and C W p (t) is the unit-volume heat capacity of water (calÆdeg )1 ÆmL )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 where D 1 0 H vh is the van’t Hoff enthalpy for transition from state 0 to state 1, D 1 0 H cal is the calorimetric enthalpy, T max is the temperature of the m aximum heat c apacity, ÆDC p æ max is theexcessheatcapacityofproteinsinthemaximumofthe peak, and D 1 0 C p is the difference between he at capacities for state 1 and 0 (after and before t he transition). CD measurements CD spectra of mutant proteins were recorded with an Aviv 62DS circular dichroism spectrometer (Aviv Inc., USA), 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 trimeric, parallel, co iled coils with a small C-terminal domain that has a b-annulus structure. In addition, we were able to obtain crystals of fibritin B1, that has 281 residues per monomer. Crystals belong to space group P2 1 , and existence o f threefold noncrystallographic symmetry pattern in observed X-ray diffraction data indicates that the B1 protein is a trimer too (N. V. Sernova, unpublished results). These data and the repetitive segmented structure of fibritin suggest that other fibritin mutants studied that have b-annulus C-terminal domain mentioned above also should have a parallel t rimeric coiled-coil structure. Indeed, all these recombinant m utants, except fibritin XN, expressed from the plasmids in E. coli cells were soluble and proteins were purified by ammonium sulfate precipitation followed by chromatography on hydroxy- apatite. Fibritin XN was refolded from inclusion bodies as described in Materials and methods. It is known that full- length fibritin, as well as some N-terminally truncated mutants, are resistant to 1% SDS [5,10,13]. These proteins do not dissociate to the monomer chains in the presence o f SDS at room temperature, and they migrate on SDS/PAGE as trimers. All the mutants used in this r esearch have such a resistance to SDS again e xcept fibritin XN ( data n ot shown) . Figure 2 shows the CD spectra of the purified fibritin mutants. These spectra indicate that all mutants, except the shortest fibritin F, exhibited properties characteristic of a high content of a helicity. The a helical contents slightly 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 NaCl. In these conditions, th e endotherm for a full-length fibritin exh ibited three well-resolved heat- absorption peaks centred at 321 K (DH ¼ 1390 kJÆmol trimer )1 ), 336 K (DH ¼ 7600 kJÆmol trimer )1 ), and 345 K (DH ¼ 515 kJÆmol trimer )1 ), respectively (Fig. 3A). The transition at 321 K can be assigned to the N-terminal region (residues 1–50), which has no heptad periodicity, and most probably to the first adjacent downstream putative coiled-coil segment (residues 51–83) and the large loop L1 (residues 8 4–96) (Fig. 1B). A ll the fibritin mutants, of different length, truncated from the N-termini had n o corresponding peak. Additionally, fibritin XN, that con- tained the N-terminus, had a heat absorption peak at 321 K of the same enthalpy as wild-type fibritin (see b elow). The transition at 345 K was clearly related to the C-terminal domain. The DSC endotherm showed that all truncated fibritin molecules, containing the C-terminal domain, had the heat absorption peak (Fig. 3A,B). Its enthalpy was approximately equal for all studied fibritin mutants (Fig. 3A, internal) as well as for the isolated C-termini [22]. The highest transition temperature of the different oligomeric protein domains was usually Table 1. Thermodynamic properties of fibritin truncated mutants. No of amino-acid 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 S1 108 )1216 )656 )560 0.81 M75 )630 – – – F58 )515 – )515 – Fig. 2. Far C D spectra of wac, B1, SM1, E , M and F fibritins. 836 S. P. Boudko et al. (Eur. J. Biochem. 269) Ó FEBS 2002 concentration dependent [22]. Indeed, t he 345 K transition of fibritin was concentration dependent (data not shown) as was found for the isolated C-termini [ 22]. In addition, the CD spectrum of fibritin SM4 indicated that the secondary structure of the C-terminal domain melts between 335 and 358 K (Fig. 4A). The DSC endotherms for B1, SM1, and SM4 mutants (all containing the C-terminal domain) revealed that the 330 K h eat adsorption t ransition was almost accomplished at 335 K, while the 345 K transition was just beginning. According to the CD data, the SM4 protein was completely unfolded at 358 K. The CD spectrum of fibritin’s C-terminal domain was calculated as the difference of spectra at 335 K and 358 K. It had a characteristic positive peak centered at 229 nm with molar ellipticity h molar ¼ 12 000 degÆcm 2 Ædmol )1 (Fig. 4 B) that was in agreement with the CD spectrum of the purified C-terminal domain [22]. The major heat absorption peak at 336 K, observed for a full-length fibritin, had an enthalpy that was four times 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 0.15 M NaCl. T he enthalpy a ssigned to t he coiled-coil part represent a lin ear dependence with the slope o f 21 kJÆmol res )1 . Fig. 3. Temperature dependence o f the partial heat capacity of fi britin mu tants i n 0 .01 M Na phosphate buffer (pH 8.0) and 0.15 M NaCl. Protein concentration was 16 m M chain )1 for the full-length fibritin, and 50 m M chain )1 for the others. (a) Thermal transition profiles of the wac, B1, SM1, SM4, M, and F m utants. (b) Thermal transition curves for the E, S1, a nd F fi britins. Ó FEBS 2002 Thermodynamics of segmented coiled coil protein (Eur. J. Biochem. 269) 837 eliminates an unusual interaction between the Asp in a d position that is mediated in fibritin E by a chloride ion located on the threefold axis [8]. This interaction, also found in other coiled-coil proteins, i s c onsidered to be important for the correct alignment of polypeptide chains upon a coiled-coil formation [23,24]. However, in fibritin, its C-terminal domain governs such an assembly alignment. Furthermore, Ile425 is well accommodated at its d position in the trimeric coiled-coil struc ture [9], and this mutation 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 DH cal for the E, SM4, SM1, B1 mutants, and for a full-length fibritin were 0.91, 0.88, 0.42, 0.39 and 0.13, respectively (Table 1), indicating a decrease of the all-or-none transition character with increasing domain size. A plot of total DH cal against the number of residues for all mutants, truncated from the N-termini, yielded a homogeneous curve with an i nitial slope of 6.5 ± 0.5 and a final slope of 27.5 ± 2 kJÆ(mol residue) )1 (Fig. 5). Preliminary results indicate that at low ionic strength (10 m M sodium phosphate buffer, pH 8.0) full-length fibr- itin exhibited two heat absorption peaks (T 1m ¼ 326 K, and T 2m ¼ 334 K) that are probably related to the transition of the coiled-coil region. The position of the 326 K peak approximately matched the position of a single transition peak of the B1, SM1, and SM4 mutants (T m 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, respectively. That bridge was initially proposed [5], but it was not found in fibritin E crystal structure [8]. Crystallo- graphic investigations of fibritin S1 structure are in progress. Refolding of the XN fibritin Due to aberrant folding, fibritin XN, lacking the C-terminal domain, was not soluble during in vivo expression and it formed aggregates [10]. We were able to purify and dissolve these aggregates in 8 M urea. Then the protein was partially refolded by the fast 100-fold dilution from 8 M to 0.08 M urea in 50 m M Tris/HCl buffer, pH 8.0 a nd purified on a hydroxyapatite column. The CD spectrum of an in vitro refolded fibritin XN was similar to the spectrum of a full- length fibritin (data not shown). However, the DSC endotherm of the refolded XN fibritin did not reveal a heat-adsorption 345 K-peak characteristic for the C-termi- nal domain, and the protein had three thermal transition peaks centred at 321 K, 329 K, and 336 K (Fig. 6A). The main d ifference between fibritin XN and other truncated fibritin molecules, which contained the C-terminal domain, was lack of ability of the XN molecule to refold after temperature-induced denaturation. After one round of heating to 340 K and subsequent slow cooling t o 293 K for 60 min, the protein revealed a complete lack of refolding (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 was completely reversible as indic ated by identical DH°-values, sharpness and h eight of the 345 K peak. DISCUSSION Previous work has demonstrated that a full-length fibritin has a complex pattern of heat-induced transitions [5] that were difficult to assign to individual domains. Also it was not possible to determine calorimetric parameters for the individual steps in transition curve and to investigate the interactions between individual segments in the three- stranded coiled-coil domain. A more detailed analysis was performed now with the help of truncated fibritin molecules. The C -terminal domain has the highest me lting temper- ature a nd it melts independently from all the other regions. Due to its trimeric nature, the midpoint temperature of the C-terminal domain transition is slightly concentration dependent, an observation which is in agreement with the results for purified domain [22]. It acts as a cross-linker between the three chains and, as it was proposed earlier [5,8,10], i t helps to align t hree chains and serves as a fo ldon by increasing local chain concentration at the C-terminus. In addition , t he C-terminal domain of fibritin, like other oligomerization domains [25,26], stabilizes adjacent upstream coiled-coil segments. For the coiled-coil region of fibritin B1, which contains about half of a fibritin sequence, only a single transition was observed. The assignment of the 330 K transition is evident from the loss of a helicity at this temperature and changes in the magnitude of the accompanying enthalpy. The ratio o f the van’t Hoff enthalpy to calorimetric enthalpy of 0.39 indicates that the nine putative segments of the coiled-coil 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 per residue in the c oiled-coil domain of all the fibritin mutants (DH res ¼ )21 JÆmol )1 ) has the same magnitude as for a three-stranded coiled-coil domain of laminin [28], and for a two-stranded coiled coil of leucine zippers [29,30]. According to CD data, we were able to refold fibritin XN, which was solved in urea, by rapid dilution. During the Fig. 6. Consequent DSC scans performed for the XN and B1 fibritin mutants in 0.01 M Na phosphate buffer (pH 8.0) with 0.15 M NaClwithascanrate of 1 KÆmin )1 . The a bsolute h eat capacity vs. th e temperatu re is shown. (a) The XN fibritin scans: t he first is of the folded fragment, the second i s after treating the fragment at 340 K for 5 min and cooling down to room t emperature for more than 1 h. (b) Consequent scans of the B1 fragment (without refilling the cells): the first two s cans were performed u ntil 336 K followed by cooling down to 298 K for 1 h; the others scans were performed until 369 K. Ó FEBS 2002 Thermodynamics of segmented coiled coil protein (Eur. J. Biochem. 269) 839 first round of DSC, the refolded XN protein exhibits several heat absorption peaks, one of which was assigned to the N-terminal domain. Following the first round of heat denaturation, it was impossible to r efold of the m olecule by slow cooling to low temperature. In contrast, full revers- ibility has been observed for all fragments containing the C-terminal domain. These results strongly confirm our previous con clusion [8,10] that the C -termini is essential for 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. REFERENCES 1. Coombs, D.H. & Eiserling, F.A. ( 1977) Studies on the structure, protein composition and assembly of the neck of bacteriophage T4. J. Mol. Biol. 116, 375–405. 2. Conley, M.P. & Wood, W.B. 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(2000) Transformation of a fragment of beta-structural bacteriophage T4 adhesin to stable alpha-helical trimer. Biochemistry (Moscow) 65, 1346–1351. 36. Krasnykh, V., Belousova, N., Korokhov, N., Mikheeva, G. & Curiel, D.T. (2001) Genetic targeting o f an a denovirus vector v ia replacement of th e fiber protei n with the phage T4 fi britin. J. Virol. 75, 4176–4183. Ó FEBS 2002 Thermodynamics of segmented coiled coil protein (Eur. J. Biochem. 269) 841 . Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein Sergei P. Boudko 1,2 , Yuri Y. Londer 1 , Andrei. mutants To investigate the stability and thermodynamic properties of T4 fibritin, a set of recombinant truncated mutants was designed and analysed. All these molecules