Engineering of deltoid and reduced deltoid: Two chimeric proteins containing the oligomerization site of the hepatitis delta antigen

Summary Hepatitis delta antigen (HDAg) must form oligomers to be biologically active. Quandrin (HDAg-(12–60)-Tyr) is a 50-residue protein segment from the oligomerization domain of HDAg. The crystal structure of quadrin shows an octamer consisting of four identical copies of a dimer containing an antiparallel α-helical coiled coil. Each end of the dimer contains an oligomerization site that interacts isologously with the oligomerization site of another dimer to form a right-angled corner. The resulting quadrin octamer is a 400-residue square protein surrounding a large aqueous hole. We have designed, chemically synthesized, and characterized deltoid and reduced deltoid, two 51-residue chimeric proteins that structurally and functionally mimic one of the two oligomerization sites of the quadrin dimer. Dimerization of deltoid or reduced deltoid should emulate the dimerization of two quadrin dimers to form one right-angled corner of the square. Deltoid and reduced deltoid were designed by molecular modeling, mechanics, and dynamics and synthesized by the solid-phase method. The amino acid sequence of deltoid (GREDILEQWVSCRKKL+PKAPPEE+LRKLKKKCKKLEEDNPWLGNIKGIIGKY) is a chimera of three protein segments: HDAg-(12–28),Thermus thermophilus serine tRNA synthase-(59–65), and HDAg-(34–60)-Tyr. Cysteine (C) was introduced at two positions to explore the effects of the presence (deltoid) or absence (reduced deltoid) of an interhelical disulfide bond. Circular dichroic spectropolarimetry revealed that both synthetic proteins from an α-helical structure that is stable over a wide range of pH and KCl concentrations. Size-exclusion chromatography indicated that deltoid and reduced deltoid each form a dimer. Interconversion of these monomers and dimers should be useful model systems for studying the structural features of the right-angled corners of the quandrin octamer that contribute to HDAg oligomerization. If, like quadrin, deltoid or reduced deltoid interferes with HDAg oligomerization, it might serve as a lead compound for the design of potent HDV inhibitors.     

Characterization of deltoid, a chimeric protein containing the oligomerization site of hepatitis delta antigen

Both forms of the hepatitis delta antigen (HDAg) encoded by hepatitis delta virus are active only as oligomers. Previous studies showed that quadrin, a synthetic 50-residue peptide containing residues 12-60 from the N-terminus of HDAg, interferes with HDAg oligomerization, forms an alpha-helical coiled coil in solution, and forms a novel square octamer in the crystal consisting of four antiparallel coiled-coil dimers joined at the corners by hydrophobic binding of oligomerization sites located at each end of the dimers. We designed and synthesized deltoid (CH3CO-[Cys23]HDAg-(12-27)-seryl-tRNA synthetae-(59-65)-[Cys42]HDAg-(34-60)-Tyr-NH2), a chimeric protein that structurally resembles one end of the quadrin dimer and contains a single oligomerization site. The 51-residue chain of deltoid contains a seven-residue alpha-hairpin loop in place of the remainder of the quadrin dimer plus Cys12 and Cys31 for forming an intrachain disulfide bridge. Reduced, unbridged deltoid (Tm=61 degrees C, DeltaG(H2O)=-1.7 kcal mol(-1)) was less stable to denaturation by heat or guanidine HCl than oxidized, intrachain disulfide-bridged deltoid (Tm>80 degrees C, DeltaG(H2O)=-2.6 kcal mol(-1)). Each form is an alpha-helical dimer that reversibly dissociates into two monomers (Kd=80 microM).     

Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen.

Two forms of hepatitis delta antigen (HDAg) have different roles in the replication cycle of hepatitis delta virus (HDV); the small forms trans activates HDV RNA replication, whereas the large form suppresses it but is needed for virion assembly. To understand the mechanism of these regulatory activities, we studied the possible HDAg oligomerization and its role in HDV replication. In this report, we provide direct biochemical evidence for the in vitro and in vivo formation of homodimers and heterodimers between these two HDAg species. By deletion mutagenesis, we showed that this protein interaction is mediated by the leucine zipper-like sequence residing in the N-terminal one-third of HDAg. Furthermore, site-specific mutants with various substitutions on two of the leucine residues in this stretch of sequence had reduced or no ability to form HDAg dimers. Correspondingly, the small HDAg with mutations in the leucine zipper-like sequence had reduced abilities to trans activate HDV RNA replication. Similar mutations on the leucine zipper-like sequence of the large HDAg also resulted in loss of the ability of large HDAg to inhibit HDV RNA replication. The in vivo biological activities of both forms of HDAg (trans activation and trans-dominant inhibition of HDV RNA replication, respectively) correlated with the extent of HDAg oligomerization in vitro. Thus, we conclude that the small HDAg participates in HDV RNA replication as an oligomer form and that the large HDAg inhibits HDV RNA replication as a result of its complex formation with small HDAg. A "black sheep" model for the mechanism of trans-dominant inhibition by the large HDAg is presented.     

The Hydrophobicity of the H3 Histone Fold differs from the Hydrophobicity of the other three Folds

The eukaryotic histone dimers, H3–H4 and H2A–H2B, are formed in the cytosol prior to being transported into the nucleus and assembled into the nucleosome. Residue side-chain distances from the interior of the histone dimers are obtained with an ellipsoidal spatial metric and structural information provided by X-ray analyses at atomic resolution of the nucleosome core particles. While the spatial hydrophobic moment profiles of the dimers are comparable with profiles obtained previously that characterize the hydrophobic core of single-chain, single-domain globular soluble proteins, correlation coefficients between the side-chain hydrophobicities and distances from the interior of the H3–H4 dimer and H2A–H2B dimer differ significantly. This difference is traced to the H3 histone fold, which segregates fewer hydrophobic residues within the protein interior than the three other folds. Examination of the correlation coefficient between residue hydrophobicity and side-chain distance from the dimer interior over local regions of the fold sequence shows that the region of reduced correlation is associated mainly with the residues at the carboxyl end of the H3 histone fold, the helical region of the fold involved in the H3–H3 binding of the (H3–H4)₂ tetramer of the nucleosome. Hydrophobic interactions apparently contribute to the binding of this fourfold helical bundle and this evolutionary requirement may trade off against the requirement for H3–H4 dimer stability. The present results provide a different view than previously proposed, albeit of similar origin, to account for the reduced stability of the H3–H4 dimer compared with the H2A–H2B dimer.Reviewing Editor: Dr. Martin Kreitman     

Local helix content and RNA-binding activity of the N-terminal leucine-repeat region of hepatitis delta antigen

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. Our results show that the N-terminal leucine-repeat region of hepatitis delta antigen (HDAg), encompassing residues 24–50, binds to the autolytic domain of HDV genomic RNA and attenuates its autolytic activity. The solution conformation of a synthetic peptide corresponding to residues 24–50 of HDAg as determined by two-dimensional ¹H NMR and circular dichroism techniques is found to be an -helix. The local helix content of this peptide was analyzed by NOEs and coupling constants. Mutagenesis studies indicate that Lys³⁸, Lys³⁹, and Lys⁴⁰ within this -helical peptide may be directly involved in RNA binding. A structural knowledge of the N-terminal leucine-repeat region of HDAg thus provides a molecular basis for understanding its role in the interaction with RNA.     

Oligomeric Properties of Survival Motor Neuron·Gemin2 Complexes

The survival motor neuron (SMN) protein forms the oligomeric core of a multiprotein complex required for the assembly of spliceosomal small nuclear ribonucleoproteins. Deletions and mutations in the SMN1 gene are associated with spinal muscular atrophy (SMA), a devastating neurodegenerative disease that is the leading heritable cause of infant mortality. Oligomerization of SMN is required for its function, and some SMA patient mutations disrupt the ability of SMN to self-associate. Here, we investigate the oligomeric nature of the SMN·Gemin2 complexes from humans and fission yeast (hSMN·Gemin2 and ySMN·Gemin2). We find that hSMN·Gemin2 forms oligomers spanning the dimer to octamer range. The YG box oligomerization domain of SMN is both necessary and sufficient to form these oligomers. ySMN·Gemin2 exists as a dimer-tetramer equilibrium with K_d = 1.0 ± 0.9 μm. A 1.9 Å crystal structure of the ySMN YG box confirms a high level of structural conservation with the human ortholog in this important region of SMN. Disulfide cross-linking experiments indicate that SMN tetramers are formed by self-association of stable, non-dissociating dimers. Thus, SMN tetramers do not form symmetric helical bundles such as those found in glycine zipper transmembrane oligomers. The dimer-tetramer nature of SMN complexes and the dimer of dimers organization of the SMN tetramer provide an important foundation for ongoing studies to understand the mechanism of SMN-assisted small nuclear ribonucleoprotein assembly and the underlying causes of SMA.     

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.     

Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

pH-, temperature- and ion-dependent oligomerization of Sulfolobus solfataricus recombinant amidase: a study with site-specific mutants

Recombinant amidase from Sulfolobus solfataricus occurred as a dimer of 110 kDa comprising identical subunits. Only dimers were present at pHs above 7.0, but with decreasing pH, dimers associated into octamers, with complete oligomerization occurring at pH 3.0. Oligomerization showed reversible temperature-dependence, with octamer formation increasing with temperature from 36 °C to between 70 and 80° C. Increasing salt concentrations, favored dissociation of the octamers. Among the three investigated factors affecting the dimer–octamer equilibrium, the most important was pH. Among four mutants obtained by site-specific mutagenesis and selection for pH and temperature sensitivity, the T319I and D487N mutant amidases, like that of the native Sulfolobus solfataricus, responded to changes in pH and temperature with a conformational change affecting the dimer–octamer equilibrium. The Y41C and L34P mutant amidases were unaffected by pH and temperature, remaining always in the dimeric state. The differences among mutants in protein conformation must be related to the position of the introduced mutation. Although the L34P and Y41C mutations are located in the helical region 33–48 (LLKLQLESYERLDSLP), which is close to the amino-terminal segment of the protein, the T319I mutation is located in a strand on the surface of the protein, which is far from, and opposite to, the amino-terminal segment. The D487N mutation is located in the center of the protein, far distant from the 33–48 segment. These observations suggest that the segment of the protein closest to the amino-terminus plays a key role in the association of dimers into octamers.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric C structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10^–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10^–6M) to 7.1 ([TPT] = 10^–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7)·10³M^–1.     

Functional characterization of the microtubule-binding and -destabilizing domains of CPAP and d-SAS-4

We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423–607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311–422). Site-specific mutagenesis revealed that the mutations that either disrupt the α-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved  20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4′s MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.     

Hydrophobized Formate Dehydrogenase from Pseudomonas sp. 101 in the System of Reverse Micelles of Aerosol OT in Octane

NAD⁺-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2–10). The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane. Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer. The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree. Thus, the modified enzyme mainly functions in the form of monomer and octamer. The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.     

Hydrogen bonding interactions in E- or Z-2-phenyl-3-( X'-pyridyl)propenoic acid ( X=2, 3 or 4) assemblies--a molecular modeling study

The agglomeration properties of stereoisomeric 2-phenyl-3-(X-pyridyl)propenoic acids (X=2, 3 or 4) were studied by the PM3 semiempirical quantum chemical method. Calculations revealed that dimers kept together by the intermolecular hydrogen bonding interactions of the carboxylic groups could be built from both stereoisomers irrespective of the position of the nitrogen heteroatom. The dimers of the Z-isomers could also be built through (aromatic)C–H...N hydrogen bonds between the dimer units. The longest agglomerate was the pentamer of the dimers when the nitrogen was in the 2 position. Longer hydrogen-bonded agglomerates than dimers could only be constructed from the E-isomer with the nitrogen in position 4. Here, the trimer of the dimers proved to be the longest hydrogen-bonded entity and similarly to the Z-isomers, the dimer units are kept together by (aromatic)C–H...N intermolecular hydrogen bonds.Figure The helical structure of the pentamer of dimers for the Z-2-phenyl-3-(2-pyridyl)propenoic acid molecule, when in the initial geometry (aromatic)C(4)–H...N close contact was enforced.     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme : A Possible Regulatory Mechanism

NAD malic enzyme can exist in dimer, tetramer, or octamer form. Freshly prepared enzyme from Solanum tuberosum var. Chieftan exists predominantly as the octamer and during storage is progressively converted into lower molecular weight forms. High ionic strength favors dimer formation, whereas high concentrations of malate or citrate favor tetramer formation. The tetramer is the most active form, having a low K_m for malate and a high V_max. The dimer, with its high K_m and low V_max, is the least active form. Malate may regulate NAD malic enzyme by controlling its state of oligomerization.     

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double β^5.7-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded β^6.3-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.     

Analysis of the dynamic equilibrium of histone oligomers in a solution. The nature of forces stabilizing the (H2A-H2B-H3-H4)2 octamer structure

Dynamic equilibrium analysis of the (H2A-H2B-H3-H4)2 histone octamer with lower oligomers was performed in 2 M NaCl. Calculated data on the relative content of histone oligomers upon changing protein concentration in solution are given. The red shift of lambda max for histone tyrosine fluorescence spectra is shown to be due to hydrogen bond formation by tyrosyl OH-groups. Analysis of free energy changes of histone oligomers upon association (delta G = -17,37 +/- 0,14 kcal/mole) as well as the effect of urea on histone octamer dissociation made it possible to conclude that virtually all tyrosyls in octamer form hydrogen bonds. Intermolecular hydrogen bonds formed by tyrosyls contribute substantially to octamer stabilization. The (H2A-H2B) dimer positive cooperativity in association with the (H3-H4)2 tetramer was found. This cooperativity is caused by interaction between association sites with a two order increase in an apparent constant of dimers with tetramer association. The histone octamer was determined to be of asymmetric structure due to unequivolency of the two binding sites for the (H2A-H2B) dimers.     

DIMERIZATION IN HIGHLY CONCENTRATED SOLUTIONS OF PHOSPHOIMIDAZOLIDE ACTIVATED MONONUCLEOTIDES

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5-monophosphate. Here we report the self-condensation of nucleoside 5-phosphate 2-methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2+ in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MeImpU and 2-MeImpC produce about 65% of oligomers including 4% of the 3,5-linked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of internucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MeImpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.     

Minimal requirements for in vitro reconstitution of the structural subunit of light-harvesting complexes of photosynthetic bacteria

Unlike the and polypeptides of the core light-harvesting complex (LH1) of Rhodobacter (Rb.) sphaeroides, the and polypeptides of the peripheral light-harvesting complex (LH2) of this organism will not form a subunit complex by in vitro reconstitution with bacteriochlorophyll. Guided by prior experiments with the LH1 polypeptides of Rb. sphaeroides and Rhodospirillum rubrum, which defined a set of interactions required to stabilize the subunit complex, a series of mutations to the Rb. sphaeroides LH2 polypeptide was prepared and studied to determine the minimal changes necessary to enable it to form a subunit-type complex. Three mutants were prepared: Arg at position –10 was changed to Asn (numbering is from the conserved His residue which is known to be coordinated to bacteriochlorophyll); Arg at position –10 and Thr at position +7 were changed to Asn and Arg, respectively; and Arg at position –10 was changed to Trp and the C-terminus from +4 to +10 was replaced with the amino acids found at the corresponding positions in the LH1 polypeptide of Rb. sphaeroides. Only this last multiple mutant polypeptide formed subunit-type complexes in vitro. Thus, the importance of the C-terminal region, which encompasses conserved residues at positions +4, +6 and +7, is confirmed. Two mutants of the LH1 polypeptide of Rb. sphaeroides were also constructed to further evaluate the interactions stabilizing the subunit complex and those necessary for oligomerization of subunits to form LH1 complexes. In one of these mutants, Trp at position –10 was changed to Arg, as found in LH2 at this position, and in the other His at position –18 was changed to Val. The results from these mutants allow us to conclude that the residue at the –10 position is unimportant in subunit formation or oligomerization, while the strictly conserved His at –18 is not required for subunit formation but is very important in oligomerization of subunits to form LH1.