Non-Structured Amino-Acid Impact on GH11 Differs from GH10 Xylanase

The Aspergillus niger xylanase (Xyn) was used as a model to investigate impacts of un-structured residues on GH11 family enzyme, because the β-jelly roll structure has five residues (Ser1Ala2Gly3Ile4Asn5) at N-terminus and two residues (Ser183Ser184) at C-terminus that do not form to helix or strand. The N- or/and C-terminal residues were respectively deleted to construct three mutants. The optimal temperatures of XynΔN, XynΔC, and XynΔNC were 46, 50, and 46°C, and the thermostabilities were 15.7, 73.9, 15.5 min at 50°C, respectively, compared to 48°C and 33.9 min for the Xyn. After kinetic analysis, the substrate-binding affinities for birch-wood xylan decreased in the order XynΔC>Xyn>XynΔNC>XynΔN, while the K_cat values increased in the order XynΔC<XynΔNC<Xyn<XynΔN. The C-terminal deletion increased the GH11 xylanase thermostability and T_opt, while the N- and NC-terminal deletions decreased its thermostability and optimal temperature. The C-terminal residues created more impact on enzyme thermal property, while the N-terminal residues created more impact on its catalytic efficiency and substrate-binding affinity. The impact of non-structured residues on GH11 xylanase was different from that of similar residues on GH10 xylanase, and the difference is attributed to structural difference between GH11 jelly-roll and GH10 (β/α)₈.     

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.     

Insights into the maturation of hyperthermophilic pyrolysin and the roles of its N-terminal propeptide and long C-terminal extension

Pyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or the maturation of these enzymes. Here, the recombinant proform of Pyrococcus furiosus pyrolysin (Pls) and several N- and C-terminal deletion mutants were successfully expressed in Escherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing of both the N-terminal propeptide and the C-terminal portion of the long CTE, indicating that the long CTE actually consists of the C-terminal propeptide and the C-terminal extension (CTEm), which remains attached to the catalytic domain in the mature enzyme. Although the N-terminal propeptide deletion mutant PlsΔN displayed weak activity, this mutant was highly susceptible to autoproteolysis and/or thermogenic hydrolysis. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of pyrolysin into its thermostable conformation. In contrast, the C-terminal propeptide deletion mutant PlsΔC199 was converted to a mature form (mPlsΔC199), which is the same size as but less stable than mPls, suggesting that the C-terminal propeptide is not essential for folding but is important for pyrolysin hyperthermostability. Characterization of the full-length (mPls) and CTEm deletion (mPlsΔC740) mature forms demonstrated that CTEm not only confers additional stability to the enzyme but also improves its catalytic efficiency for both proteineous and small synthetic peptide substrates. Our results may provide important clues about the roles of propeptides and CTEs in the adaptation of hyperthermophilic proteases to hyperthermal environments.     

Hepatitis B virus: DNA polymerase activity of deletion mutants

The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.     

The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

Suppression of a frameshift mutation in the recE gene of Escherichia coli K-12 occurs by gene fusion.

The nucleotide sequences of a small gene, racC, and the adjacent N-terminal half of the wild-type recE gene are presented. A frameshift mutation, recE939, inactivating recE and preventing synthesis of the active recE enzyme, exonuclease VIII, was identified. The endpoints of five deletion mutations suppressing recE939 were sequenced. All five delete the frameshift site. Two are intra-recE deletions and fuse the N- and C-terminal portions of recE in frame. Three of the deletions remove the entire N-terminal portion of recE, fusing the C-terminal portion to N-terminal portions of racC in frame. These data indicate that about 70% of the N-terminal half of recE is not required to encode a hypothesized protein domain with exonuclease VIII activity.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2

To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (α-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 °C that was 10 °C higher than the native Xyn2. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA.     

The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.     

Xyn11A, a multidomain multicatalytic enzyme fromPseudobutyrivibrio xylanivorans Mz5T

The rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T has a potent xylanolytic enzyme system. A small native peptide (approximately 30-kDa, designated Xyn11A) from the bacterium was first isolated and characterized by Edman degradation. The gene coding for Xyn11A was identified using PCR amplification with consensus primers. It was then fully sequenced to reveal an open reading frame of 1809 bp. The predicted N-terminal domain exhibited xylanolytic activity and was classed to the family 11 of glycosyl hydrolases; it is followed by a region with homology to a family 6 cellulose binding module. The C-terminal domain codes for a putative NodB-like polysaccharide deacetylase which is predicted to be an acetyl esterase implicated in debranching activity in the xylan backbone. As similar domain organization was also found in several other xylanases from a diverse range of bacteria, a common ancestor of such a xylanase is considered to be present and spread, possibly by horizontal gene transfer, to other microorganisms from different ecological niches.     

Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase

We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa. Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp. strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5. xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence. Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains. Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101. Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa. Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer. Thus, Paenibacillus sp. strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains. Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.     

Circular permutation and deletion studies of myoglobin indicate that the correct position of its N-terminus is required for native stability and solubility but not for native-like heme binding and folding

We studied the effect of deleted and circularly permuted mutations in sperm whale myoglobin and present here results on three classes of mutants: (i) a deletion mutant, Mb(1)(-)(99), in which the C-terminal helices, G and H, were removed; (ii) two circular permutations, Mb-B_GHA, in which helix B is N-terminal and helix A is C-terminal, and Mb-C_GHAB, in which helix C is N-terminal and helices A and B are C-terminal; and (iii) a deleted circular permutation, Mb-HAB_F, in which helix H is N-terminal, helix F is C-terminal, and helix G is deleted. The conformational characteristics of the apo and holo forms of these mutants were determined at neutral pH, by spectroscopic and hydrodynamic methods. The apo form of the deleted and permuted mutants exhibited a stronger tendency to aggregate and had lower ellipticity than the wild type. The mutants retained the ability to bind heme, but only the circularly permuted holoproteins had native-like heme binding and folding. These results agree with the theory that myoglobin has a central core that is able to bind heme, but also indicate that the presence of N- and C-terminal helices is necessary for native-like heme pocket formation. Because the holopermuteins were less stable than the wild-type protein and aggregated, we propose that the native position of the N-terminus is important for the precise structural architecture of myoglobin.     

Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module

Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)(8) barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.     

Effects of dockerin domains on Neocallimastix frontalis xylanases

Two xylanase genes were cloned from the anaerobic fungus Neocallimastix frontalis. Xyn11A had a modular structure of two catalytic domains and two dockerin domains, while Xyn11B had one catalytic domain and two dockerin domains. The characteristics of the xylanases with and without dockerin domains were investigated. The deletion of dockerin domains had little influence on the optimal pH of xylanases, while it significantly affected the optimal temperatures. The optimal temperatures increased from 55 to 60 degrees C for Xyn11A and 60 to 65 degrees C for Xyn11B after the deletion of dockerin domains. The increase of optimal temperatures was attributed to the lower stability of the second structure in full length xylanase than that in the truncated one as evidenced by the circular dichroism spectroscopy. The specific activity of Xyn11A and Xyn11B increased about 64% and 330%, respectively, after the deletion of the dockerin domains. The removal of dockerin domains appeared to increase the overall efficiency of Xyn11A' (1.2-) and Xyn11B' (2.9-) fold with oat spelts xylan as reflected by the values of k(cat)/K(m). The results suggest that the dockerin domain might play an important role in the characteristics of xylanases from anaerobic fungi.     

The N-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum is not a thermostabilizing domain

Xylanase Xyn10B from Clostridium thermocellum is a modular enzyme that contains two family 22 carbohydrate binding modules N- (CBM22-1) and C- (CBM22-2) terminal of the family 10 glycoside hydrolase catalytic domain (GH10). In a previous study, we showed that removal of CBM22-1 reduces the resistance to thermoinactivation of the enzyme suggesting that this module is a thermostabilizing domain. Here, we show that it is the module border on the N-terminal side of GH10 that confers resistance to thermoinactivation and to proteolysis. Therefore, CBM22-1 does not function as a thermostabilizing domain and the role for this apparently non-functional CBM remains elusive.     

The highly processive DNA polymerase of bacteriophage T5. Role of the unique N and C termini

The DNA polymerase encoded by bacteriophage T5 has been reported previously to be processive and to catalyze extensive strand displacement synthesis. The enzyme, purified from phage-infected cells, did not require accessory proteins for these activities. Although T5 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I and T7 DNA polymerase, it contains unique regions of 130 and 71 residues at its N and C termini, respectively. We cloned the gene encoding wild-type T5 DNA polymerase and characterized the overproduced protein. We also examined the effect of N- and C-terminal deletions on processivity and strand displacement synthesis. T5 DNA polymerase lacking its N-terminal 30 residues resembled the wild-type enzyme albeit with a 2-fold reduction in polymerase activity. Deletion of 24 residues at the C terminus resulted in a 30-fold reduction in polymerase activity on primed circular DNA, had dramatically reduced processivity, and was unable to carry out strand displacement synthesis. Deletion of 63 residues at the C terminus resulted in a 20,000-fold reduction in polymerase activity. The 3' to 5' double-stranded DNA exonuclease activity associated with T5 DNA polymerase was reduced by a factor of 5 in the polymerase truncated at the N terminus but was stimulated by a factor of 7 in the polymerase truncated at the C terminus. We propose a model in which the C terminus increases the affinity of the DNA for the polymerase active site, thus increasing processivity and decreasing the accessibility of the DNA to the exonuclease active site.     

Characterization of telomere-binding activity of replication factor C large subunit p140.

The large subunit of RFC (RFC p140) has been suggested to be associated with the 3'-end of elongating DNA primer and to recruit proliferating cell nuclear antigen (PCNA) onto DNA polymerase delta. Previously, we isolated a cDNA clone encoding a DNA-binding domain of RFC p140 as a telomeric repeat (TTAGGG)n binding protein. This domain was shown to have a specific affinity for the 5'-phosphate ends of a telomere repeat sequence. In order to investigate the structure and function of RFC p140, we constructed the full-length recombinant RFC p140 as well as N- and/or C-terminal deleted mutants and analyzed their telomere-binding activities. South-Western blot and gel mobility shift analyses revealed that deletion of the N- but not the C-terminal region enhances recognition of the telomeric repeat sequence and 5'-phosphate ends, suggesting the negative effect of the N-terminal region of the RFC p140 binding to the telomeric repeat. On the other hand, the C-terminal truncated RFC inhibits the telomerase activity more than the N-terminal-deleted and full-length RFC p140. The inhibitory effect of RFC p140 on telomerase activity is completely diminished by both terminal deletions. Thus, a certain interaction of the N- and C-terminal regions is considered to be required for RFC p140 to suppress telomerase activity. Taken together, these results suggest that both telomeric repeat-binding and telomerase inhibitory activities of RFC p140 are finely regulated by the intrinsic N- and C-terminal regions.     

Influence of N-terminal helix bundle stability on the lipid-binding properties of human apolipoprotein A-I

As the principal component of high-density lipoprotein (HDL), apolipoprotein (apo) A-I plays essential roles in lipid transport and metabolism. Because of its intrinsic conformational plasticity and flexibility, the molecular details of the tertiary structure of lipid-free apoA-I have not been fully elucidated. Previously, we demonstrated that the stability of the N-terminal helix bundle structure is modulated by proline substitution at the most hydrophobic region (residues around Y18) in the N-terminal domain. Here we examine the effect of proline substitution at S55 located in another relatively hydrophobic region compared to most of the helix bundle domain to elucidate the influences on the helix bundle structure and lipid interaction. Fluorescence measurements revealed that the S55P mutation had a modest effect on the stability of the bundle structure, indicating that residues around S55 are not pivotally involved in the helix bundle formation, in contrast to the insertion of proline at position 18. Although truncation of the C-terminal domain (Δ190-243) diminishes the lipid binding of apoA-I molecule, the mutation S55P in addition to the C-terminal truncation (S55P/Δ190-243) restored the lipid binding, suggesting that the S55P mutation causes a partial unfolding of the helix bundle to facilitate lipid binding. Furthermore, additional proline substitution at Y18 (Y18P/S55P/Δ190-243), which leads to a drastic unfolding of the helix bundle structure, yielded a greater lipid binding ability. Thus, proline substitutions in the N-terminal domain of apoA-I that destabilized the helix bundle promoted lipid solubilization. These results suggest that not only the hydrophobic C-terminal helical domain but also the stability of the N-terminal helix bundle in apoA-I are important modulators of the spontaneous solubilization of membrane lipids by apoA-I, a process that leads to the generation of nascent HDL particles.