Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.     

A single Gly114Arg mutation stabilizes the hexameric subunit assembly and changes the substrate specificity of halo-archaeal nucleoside diphosphate kinase

Nucleoside diphosphate kinase from extremely halophilic archaeon (HsNDK) requires above 2M NaCl concentration for in vitro refolding. Here an attempt was made to isolate mutations that allow HsNDK to refold in low salt media. Such a screening resulted in isolation of an HsNDK mutant, Gly114Arg, which efficiently refolded in the presence of 1M NaCl. This mutant, unlike the wild type enzyme, was expressed in Escherichia coli as an active form. The residue 114 is in close proximity to Glu155 of the neighboring subunit in the three dimensional hexameric structure of the HsNDK. It is thus possible that the attractive electrostatic interactions occur between Arg114 and Glu155 in the mutant HsNDK, stabilizing the hexameric subunit assembly.     

Molecular Mechanism of Distinct Salt-Dependent Enzyme Activity of Two Halophilic Nucleoside Diphosphate Kinases

Nucleoside diphosphate kinases from haloarchaea Haloarcula quadrata (NDK-q) and H. sinaiiensis (NDK-s) are identical except for one out of 154 residues, i.e., Arg³¹ in NDK-q and Cys³¹ in NDK-s. However, the salt-dependent activity profiles of NDK-q and NDK-s are quite different: the optimal NaCl concentrations of NDK-q and NDK-s are 1 M and 2 M, respectively. We analyzed the relationships of the secondary, tertiary, and quaternary structures and NDK activity of these NDKs at various salt concentrations, and revealed that 1), NDK-q is present as a hexamer under a wide range of salt concentrations (0.2-4 M NaCl), whereas NDK-s is present as a hexamer at an NaCl concentration above 2 M and as a dimer at NaCl concentrations below 1 M; 2), dimeric NDK-s has lower activity than hexameric NDK-s; and 3), dimeric NDK-s has higher helicity than hexameric NDK-s. We also determined the crystal structure of hexameric NDK-q, and revealed that Arg³¹ plays an important role in stabilizing the hexamer. Thus the substitution of Arg (as in NDK-q) to Cys (as in NDK-s) at position 31 destabilizes the hexameric assembly, and causes dissociation to less active dimers at low salt concentrations.     

Effects of salinity and nitric oxide donor sodium nitroprusside (SNP) on development and salt secretion of salt glands of Limonium bicolor

NO, as a signaling molecule, is involved in abiotic stresses. Limonium bicolor seedlings were treated with 200 mM NaCl combined with 0.05 mM SNP for 20 days to study the effects of NO on development and salt-secretion rates of salt glands. It was shown that the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased significantly compared with that under 200 mM NaCl treatment. Na⁺ secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was significantly higher than that under 200 mM NaCl without SNP. However, there was no significant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. Although there was no significant difference in salt-secretion rate of individual glands, Na⁺ concentration in the leaves treated with 200 mM NaCl solution containing SNP was significantly lower than that treated with 200 mM NaCl solution. Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na⁺ concentration in salt glands. Obviously, the efficiency of the secretion process per gland was enhanced by adding SNP to NaCl. The results showed NO may enhance the salt secretion by inducing more dermatogen cells to develop into salt glands and by enhancing the efficiency of the secretion process per gland.     

Effects of salinity and clipping on biomass and competition between a halophyte and a glycophyte

Global climate change will likely result in the reduction of water levels in intermountain wetlands and ponds, and the vegetation communities associated with these wetlands are an important forage source for livestock. Lowered water levels will not only constrict wetland plant communities, it will potentially change aquatic and soil salt concentrations. Such an increase in salinity can reduce plant growth and potentially affect competitive interactions between plants. A greenhouse experiment examined the effects of salinity and competition on the growth of two wet meadow grass species, Poa pratensis (a glycophyte) and Puccinellia nuttalliana (a halophyte). The following hypotheses based on published data were tested: (1) Biomass of both species will decrease with increasing concentration of salt; (2) root:shoot (R:S) ratio of P. pratensis will decrease with increasing salt concentration while R:S ratio of P. pratensis and P. nuttalliana will increase with clipping; (3) competitive importance will decrease for P. pratensis and P. nuttalliana with increasing salt concentration because salt induces a stress response and competitive importance is reduced in stressed environments. A factorial design included 3 plant treatments (P. nuttalliana alone, P. pratentsis alone, P. nuttalliana + P. pratensis) × 4 salinity rates (control; 5, 10, 15 g/L NaCl) × 2 clipping intensities (plants clipped or not clipped) for a total of 24 combinations replicated 6 times over a period of 90 days. We found a reduction in dry biomass as salinity increased, and this effect was greatest for P. pratensis. (1.94 g (SE 0.13) at 0 g/L NaCl to 0.22 g (SE 0.11) at 15 g/L NaCl). The R:S ratio of P. pratensis was reduced by salinity, but not for P. nuttalliana. Competitive importance of both species was reduced by clipping and by salinity, but the effect was greater and more consistent for P. pratensis. We conclude that salt concentration reduces plant growth and the effect of competition.     

Insights into the molecular relationships between malate and lactate dehydrogenases: structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric L-[LDH-like] malate dehydrogenase from the halophilic archaeon Haloarcula marismortui

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dimeric, and tetrameric species were thus characterized. The properties of the dimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs. A detailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanation of why some can form tetramers and others cannot. The study presented here makes Hm MalDH the best characterized example so far of an LDH-like MalDH.     

Interactions across the interface contribute the stability of homodimeric 3α-hydroxysteroid dehydrogenase/carbonyl reductase

The dimerization of 3α-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 °C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3α-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.     

Effect of salt stress on growth parameters, enzymatic antioxidant system, and lipid peroxidation in wild chicory (Cichorium intybus L.)

Efficient utilization of saline land for food cultivation can increase agricultural productivity and rural income. To obtain information on the salt tolerance/susceptibility of wild chicory (Cichorium intybus L.), the influence of salinity (0–260 mM NaCl) on chicory seed germination and that of two salinity levels of irrigation water (100 and 200 mM NaCl) on plant growth, antioxidative enzyme activity, and accumulation of proline and malondialdehyde (MDA) were investigated. The trials were performed outdoors, in pots placed under a protective glass covering, for two consecutive years. Seeds showed a high capacity to germinate in saline conditions. The use of 100 mM NaCl solution resulted in 81 % germination, whereas seed germinability decreased below 40 % using salt concentrations above 200 mM NaCl. Wild chicory showed tolerance to medium salinity (100 mM NaCl), whereas a drastic reduction in biomass was observed when 200 mM NaCl solution was used for irrigation. MDA, present in higher amounts in leaves than in roots, decreased in both tissues under increasing salinity. Proline content increased remarkably with the level of salt stress, more so in roots than in leaves. In salt stress conditions, the activity of antioxidant enzymes (APX, CAT, POD, SOD) was enhanced. The electrophoretic patterns of the studied enzymes showed that the salinity of irrigation water affected only the intensity of bands, but did not activate new isoforms. Our results suggest that wild chicory is able to grow in soil with moderate salinity by activating antioxidative responses both in roots and leaves.     

Noncovalent interactions of the Apple 4 domain that mediate coagulation factor XI homodimerization

The Apple 4 (A4) domain of human plasma factor XI (FXI) was used to investigate the process of FXI noncovalent dimer formation. Recombinant 6-histidine-tagged A4 domain proteins were prepared utilizing a bacterial expression system. Purification was accomplished under denaturing conditions, followed by a refolding protocol to facilitate correct disulfide bond formation. Analysis of the A4 domain (C321S mutant) by size exclusion chromatography indicated the presence of a slowly equilibrating reversible monomer-dimer equilibrium. The elution profiles reveal highly symmetrical peaks for both dimeric and monomeric species with elution times that were highly reproducible for varying amounts of both the dimeric and monomeric species. The monomer-dimer equilibrium was found to be dependent upon changes in both pH and salt concentration. Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by a dissociation constant (K(D)) value of 229 +/- 26 nm with a calculated Delta G value of 9.1 kcal/mol. This report identifies electrostatic contributions and the presence of a hydrophobic component that mediate interactions at the A4 domain interface. The rate of dissociation for the recombinant A4 domain C321S mutant was examined by monitoring the increase in 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt fluorescence under dissociating conditions, giving a value for a dissociation rate constant (k(off)) of 4.3 x 10(-3) s(-1).     

Pressure-induced subunit dissociation and unfolding of dimeric beta-lactoglobulin.

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.     

Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S.

In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD(+) but is catalytically inactive. The mutant proteins were denatured separately in 4 M urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mM NAD(+), 1 M fluoride and artificial chaperones (4 mM polyoxyethylene 10 lauryl ether and 1.6 mM beta-cyclodextrin). Under these conditions approximately 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion-exchange chromatography. Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which binds close to the NAD(+) binding site, thus abolishing coenzyme binding in the wild-type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye-ligand affinity chromatography on a Procion Red HE-3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB-modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis-Menten kinetics when the NAD(+) concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that 'nonlinear' behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.     

Salinity improves chilling resistance in Suaeda salsa

Suaeda salsa L., a C3 euhalophytic herb, is native to saline soils, demonstrates high resistance to salinity stress. The effect of chilling stress on S. salsa under high salinity, particularly the change in unsaturated fatty acid content within membrane lipids, has not been investigated. After a 12 h chilling treatment (4 °C) performed under low irradiance (100 μmol m^?2 s^?1), the chlorophyll contents, maximal photochemical efficiency of photosystem II (F _v/F _m) and actual PSII efficiency (ΦPSII) were determined. These measurements were significantly decreased in S. salsa leaves in the absence of salt treatment yet there were no significant changes with a 200 mM NaCl treatment. Chlorophyll contents, F _v/F _m and ΦPSII in S. salsa under 200 mM NaCl were higher than those without salt treatment. The unsaturated fatty acid content and the double bond index (DBI) of major membrane lipids of monogalactosyldiacylglycerols, digalactosyldiacylglycerols (DGDG), sulphoquinovosyldiacylglycerols and phosphatidylglycerols (PG) significantly increased following the chilling treatment (4 °C) (with 12 h of low irradiance and 200 mM of NaCl). The DBI of DGDG and PG was decreased in the absence of the salt treatment. These results suggest that in the euhalophyte S. salsa, a 200 mM NaCl treatment increases chilling tolerance under conditions of low irradiance (100 μmol m^?2 s^?1).     

Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg⁸⁰-Asp⁹³ as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.     

A novel surfactant-, NaCl-, and protease-tolerant β-mannanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. HJ14

A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0–30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0–30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5–2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.     

Denaturation of bovine liver glutamate dehydrogenase by guanidine hydrochloride. Correlation between enzymatic activity and molecular state

Bovine liver glutamate dehydrogenase (GDH), a hexameric enzyme, undergoes subunit dissociation, denaturation, and inactivation in the presence of guanidine hydrochloride (GdnHCl), depending on the denaturant concentration. The correlation between the enzymatic activity and the molecular state of GDH, and the reconstitution of native hexamer from subunits after the removal of GdnHCl were examined by measuring the enzymatic activity and CD spectrum in the far ultraviolet region. It was found that only the hexameric form of GDH has enzymatic activity, and the reconstitution of the hexamer with full enzymatic activity from the trimeric form which has native polypeptide chain structure can be achieved by the removal of GdnHCl. On the other hand, the recovery of enzymatic activity from the dissociated form in more concentrated GdnHCl solution where unfolding of the polypeptide chain takes place showed an exponential decrease with increasing incubation time in the GdnHCl solution. The time constant for the decay of enzymatic activity with respect to the incubation time was almost the same as that for unfolding of the polypeptide chain (followed by CD spectroscopy). It is suggested on the basis of these experimental results that the failure of reconstitution of GDH hexamer from subunits produced at high denaturant concentration is due to failure in the refolding of the unfolded subunit to the correct three-dimensional structure of the polypeptide chain rather than in the reassociation process from subunits.     

The Effects of One Amino Acid Substitutions at the C-Terminal Region of Thermostable L2 Lipase by Computational and Experimental Approach

The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris–HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris–HCl pH 8, while for L2 lipase it was at 70 °C in Glycine–NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T _m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.     

Killing of target cells by redirected granzyme B in the absence of perforin

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 °C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.     

Analysis of an N-terminal deletion in subunit <Emphasis Type="Italic">a</Emphasis> of the <Emphasis Type="Italic">Escherichia coli</Emphasis> ATP synthase

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6–20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.