Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.     

Effects of Precise Deletions in Rhodobacter sphaeroides Reaction Center Genes on Steady-state Levels of Reaction Center Proteins: A Revised Model for Reaction Center Assembly

Possible interactions between photosynthetic reaction center (RC) proteins that protect these membrane proteins from proteolytic digestion in RC complex assembly were evaluated by use of translationally in-frame (nonpolar) RC gene-specific deletions. The RC H, RC M and RC L proteins were produced from plasmids, either alone or in concert with one or both of the others, in a strain of Rhodobacter sphaeroides that contained chromosomal deletions of all three RC genes. The steady-state amounts of these proteins in cell membrane and soluble fractions were assessed in western blots. The data are used to propose a model of RC assembly in which the RC M protein accumulates in the cell membrane regardless of the presence of the RC H and RC L proteins, and the RC M protein is a nucleus for addition of RC L followed by RC H in assembly of the RC holocomplex.     

Contributions of immunoglobulin heavy and light chains to antibody specificity for lysozyme and two haptens

Chain recombination experiments with a set of structurally and/or functionally related antibodies were performed to assess the role of the heavy (H) and light (L) chains in determining antigen specificity. The results demonstrated that specificity for hen egg white lysozyme vs two haptens (dinitrophenyl or galactan) is H chain determined and for one set of proteins could be attributed specifically to the H3 region. In contrast to hapten vs lysozyme specificity, when reassociated molecules derived from structurally unrelated antibodies that bound nonoverlapping epitopes on lysozyme were tested, localization of binding to a particular epitope on lysozyme could be predominated by either H or L chains. Furthermore, in some cases, unique specificities distinct from those of either parental antibody were formed. Replacement of the native L chain with an isotypically homologous L chain was more likely to restore high affinity protein binding than was replacement of a less related L chain. When isotypically homologous L chains were compared in association with the same H chain, fine specificity profiles were sensitive to substitutions in as few as two residues that could be attributed to somatic mutation. These results demonstrate that both affinity and specificity derive from very subtle interactions between H and L chains and provide examples of how VH assembly, VL-VH pairing, and somatic mutation could contribute to development and maturation of the specificity repertoire.     

J chain in Rana catesbeiana high molecular weight Ig

A polypeptide homologous to human and mouse J chain has been identified in the high molecular weight (HMW) Ig of the bullfrog, Rana catesbeiana. In previous studies, we had detected a component that was similar in size to mammalian J chains and that, relative to L chains, migrated rapidly to the anode in alkaline-urea PAGE; however, its mobility was less than that of mammalian J chains. We now demonstrate that this component is covalently linked to the H chain of R. catesbeiana HMW Ig. All of the disulfide bridges of this polypeptide, like those of human and mouse J chain, can be cleaved by reducing agents even in the absence of denaturing solvents. The putative frog J chain was isolated by a procedure that did not require preliminary purification of the HMW Ig. The chain differed in amino acid composition from L chains but resembled J chains from several other species. Tryptic peptides were isolated and sequenced. Except for a single heptapeptide, the peptides could be aligned by virtue of their similarity to segments of human and mouse J chain. Of the 116 residues that were placed, 55 were identical with residues in human J chain and 60 with residues in mouse J chain. The six cysteine residues identified in the frog J chain are at the same positions as six of the eight cysteines in the human and mouse J chains. The results indicate significant conservation in structure between amphibian and mammalian Ig J chains.     

Quantum Chemical Studies on Proteins in the Reaction Center of Rhodobacter sphaeroides

The electronic structure of protein chains L and M in photosynthetic reaction center (PRC) of Rhodobacter sphaeroides (Van Niel) Imhoff, Truper et Pfennig) was studied by using the Overlapping Dimer Approximation method and the Extended Negative Factor Counter method at ab initio level. The result indicated that: (1) Amino acid residues, the molecular orbitals of which composed the main components of frontier orbitals of protein chain L (M ), are located at the random coil areas of chain L (α helix areas of chain M ). Since the random coil is flexible and more easy to change its conformation in the electron transfer process and to reduce the energy of the system, and the structure of the α helix is reletively stable, this difference might be one of the causes for the electron transfer in photosynthetic reaction center (PRC) only takes place along the L branch. (2) The His residues which axially coordinated to the “special pair” P and accessory chlorophyll molecules （ABChls） are essentially important for the ELUMO levels of P and ABChl. But, the corresponding molecular orbitals of these His residues do not appear in the composition of frontier orbitals of protein chains. It means that the interaction between pigment molecules and protein chains do not influence the contribution to the frontier orbitals of protein chains explicitly, but influences the corresponding ELUMO levels significantly.     

Crystal structure of a secreted insect ferritin reveals a symmetrical arrangement of heavy and light chains

Ferritins are iron storage proteins made of 24 subunits forming a hollow spherical shell. Vertebrate ferritins contain varying ratios of heavy (H) and light (L) chains; however, known ferritin structures include only one type of chain and have octahedral symmetry. Here, we report the 1.9A structure of a secreted insect ferritin from Trichoplusia ni, which reveals equal numbers of H and L chains arranged with tetrahedral symmetry. The H/L-chain interface includes complementary features responsible for ordered assembly of the subunits. The H chain contains a ferroxidase active site resembling that of vertebrate H chains with an endogenous, bound iron atom. The L chain lacks the residues that form a putative iron core nucleation site in vertebrate L chains. Instead, a possible nucleation site is observed at the L chain 3-fold pore. The structure also reveals inter- and intrasubunit disulfide bonds, mostly in the extended N-terminal regions unique to insect ferritins. The symmetrical arrangement of H and L chains and the disulfide crosslinks reflect adaptations of insect ferritin to its role as a secreted protein.     

Structure of the detergent phase and protein-detergent interactions in crystals of the wild-type (strain Y) Rhodobacter sphaeroides photochemical reaction center

Rhodobacter sphaeroides (strain Y) reaction center (RC) crystals were grown in the presence of n-octyl beta-glucoside (beta-OG). In order to determine the structure of the detergent phase in these crystals, low-resolution neutron diffraction experiments were performed at different contrasts obtained by varying the H2O/D2O ratio in the solvent. From the contrast variation data and from the RC atomic coordinates determined by X-ray diffraction [Arnoux, B., Ducruix, A., Reiss-Husson, F., Lutz, M., Norris, J., Schiffer, M., & Chang, C. H. (1989) FEBS Lett. 258, 47-50], a model was obtained for the structure of the detergent phase in the crystal. The detergent forms a ring-shaped micelle surrounding the most hydrophobic part of the transmembrane alpha helices of the RC. Each detergent ring is connected to two next-neighbor rings by intermicellar bridges. The detergent phase is organized thus in infinite zigzag chains parallel to the b axis of the P2(1)2(1)2(1) unit cell. The main interactions between beta-OG molecules and the RC molecules are hydrophobic and are localized at the level of the transmembrane alpha helices. This interaction replaces the phospholipid-protein interaction existing in vivo in the membrane and, to some extent, also the light harvesting complex-protein interaction. Secondary hydrophilic interactions are found between a few of the charged residues of the H subunit and the hydrophilic surface of the detergent ring from a neighboring RC molecule. A comparison with a previous study on Rhodopseudomonas viridis crystals [which grow in the presence of lauryldimethylamine N-oxide (LDAO) and belong to a different space group] [Roth, M., Lewit-Bentley, A., Michel, H., Deisenhofer, J., Huber, R., & Oesterhelt, D. (1989) Nature 340, 659-661] shows a quasi identity of shape and position of the beta-OG and LDAO rings around the transmembrane alpha helices. The secondary interactions, involving in both cases the external surface of the H subunit, differ because of the different molecular packing in the two space groups. The role and structural requirements of the detergent in the crystallization process are discussed.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.     

Unusual joining sites in the H and L chains of an anti-lysozyme antibody

Nucleotide sequences of HyHEL-5, an antibody specific for chicken lysozyme (HEL), indicated unusual joins in the third complementarity-determining region of both the H and L chains. The VK-JK recombination site is unusual in that codon 96, normally derived from the JK gene segment, is deleted entirely, making the L3 one amino acid shorter than normal. Examination of the HyHEL-5 Fab-HEL x-ray structure suggests that the conformation of L3 is clearly important for Ag specificity. A comparison of the HyHEL-5 L3 with that of the structurally related antibody J539 indicates that the deleted residue significantly alters the conformation of the L3 turn. The H chain VH-DH join is also unusual; the VH junction site has probably occurred between the second and third nucleotides of codon 92, with the addition of five random nucleotides that encode for unusual amino acids Leu93 and His94. Although the conformation of H3 is different from what would be predicted from other H3 conformations and is clearly important to the complementarity of HyHEL-5 to HEL, the specific residues at the VH-DH join do not appear to directly contribute to Ag binding. It is not possible to attribute the main chain conformation of H3 to the particular sequence produced by the join; the structural features of H3 may be due to interactions with HEL and/or with other antibody residues.     

Effects of photosynthetic reaction center H protein domain mutations on photosynthetic properties and reaction center assembly in Rhodobacter sphaeroides

Purple bacterial photosynthetic reaction center (RC) H proteins comprise three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane alpha-helix; and a large C-terminal, globular cytoplasmic domain. We studied the roles of these domains in Rhodobacter sphaeroides RC function and assembly, using a mutagenesis approach that included domain swapping with Blastochloris viridis RC H segments and a periplasmic domain deletion. All mutations that affected photosynthesis reduced the amount of the RC complex. The RC H periplasmic domain is shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RC M. The RC H cytoplasmic domain also functions in RC complex assembly. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RC M is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RC M and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. We suggest that RC M provides a nucleus for RC complex assembly, and that a RC H/M/L assemblage results in a cytoplasmic pool of soluble RC M and RC L proteins to provide precursors for maximal production of the RC complex.     

Characterization of the combining site of mouse myeloma protein M315

The interaction of M315 with 2,4-dinitrophenyl haptens was studied. 2,4-Dinitroaniline (DNP-NH2) showed maximum affinity to M315 at about pH 4. The pH dependence of the association constant of DNP-NH2 to M315 showed three transitions at pH 4.7, at pH 7.2, and below pH 9, respectively. Since the DNP-NH2 molecule has no charged group in this pH range, the transitions were explained in terms of amino acid residues with ionizable side chains in M315. Judging from the pK values and the effect of succinylation, these transitions were concluded to be related to ionizations of carboxyl, imidazole, and phenol groups, respectively. Measurement of the fluorescence of affinity-labeled M315 suggested that the transition at pH 4.7 reflected an equilibrium between two forms of M315 with different conformations of the combining site. The contribution of the amino acid sequence on the light (L) chain to the interaction with haptens was studied by use of antibodies (Abs) reconstituted from the heavy chain of M315 (H315) and either a homologous or a heterologous L chain. The reconstituted heterologous Ab (H315L952) showed similar pH dependence of binding to DNP-NH2 to that of the homologous Ab (H315L315). Moreover, the two Abs showed no appreciable difference in binding to DNP-haptens of different sizes. These results suggested that the difference in the amino acid sequences of L315 and L952, which originated by a somatic hypermutation, has little effect on the ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

A possible difference in the heterogeneity of the heavy and light chains from a rabbit anti-p-azobenzoate antibody preparation.

CNBr cleavage of rabbit heavy (H) chains leads to the formation of a fragment, C-1, which consists of the N-terminal half of the H chain. Fragment C-1 is cleaved at methionyl residues but held together by intrachain S-S bonds so that smaller fragments can be liberated by total reduction and alkylation. In the case of the C-1 fragment from an anti-p-azobenzoate antibody preparation, which has a light (L) chain of markedly restricted heterogeneity, total reduction and alkylation liberated seven major fragments in good yield. The N-terminus of two of these fragments corresponds to position 35 of the H chain but their N-terminal sequences are clearly different. The H chain regions represented by the other fragments implied that they were derived from H chains having different distributions of methionyl residues. This hypothesis was supported by isolating six different antibody components from the antibody preparation by isoelectric focusing and then digesting them with CNBr. Comparison of the products showed that the six components all appeared to behave differently. These results are interpreted as suggesting that the process whereby H and L chains are paired in vivo may not be completely specific and may provide a simple means of generating a significant contribution to antibody diversity.     

Botulinum neurotoxin type A radiolabeled at either the light or the heavy chain

Botulinum neurotoxin (NT) has two distinct structural regions called L and H chains (approximately 50 and approximately 100 kDa, respectively). Although the H chain is responsible for binding of the NT to neuronal cells, it is not known which of the subunits is internalized and therefore responsible for causing the blockage of acetylcholine release in susceptible neuronal cells. In this report we describe for the first time the preparation of type A NT which is selectively radiolabeled at either the L or the H chain subunit. Such NT preparations will be useful as tools for determining the distribution of L and H chains in poisoned neuronal cells and the role that each subunit plays in inducing toxicity. The L and H chains of the NT (approximately 150 kDa) were separated, purified, and then individually radiolabeled by reductive methylation of the lysine residues using [3H]- or [14C]formaldehyde. The labeled L and H chains were reconjugated with the complementary unlabeled L and H chains. Formation of -S-S- and noncovalent bonds between the L and H chains regenerated the approximately 150 kDa NT. Autoradiographs of sodium dodecyl sulfate polyacrylamide gels confirmed that each reconstituted NT preparation was labeled at only one subunit chain. NT selectively labeled at either the L or the H chain had specific radioactivities of ca. 25-30 and 45-55 microCi/mumol, respectively, and toxicity (mouse LD50/mg protein) values of 2.2 +/- 1.1 X 10(7) and 3.0 +/- 1.0 X 10(7), respectively. A linear increase in the specific radioactivity of L and H chain subunits was observed with increasing concentrations of 3H- or 14C-labeled formaldehyde in the reaction mixture and with increasing concentrations of L or H chain in the reaction mixture.     

Replacement of a conserved arginine in the assembly domain of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation.

In higher plants the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase, EC 4.1.1.39) contains a segment of 16 amino acids which is absent from cyanobacterial S. This segment connecting two beta sheets has been shown, by crystallographic analysis, to form a hairpin loop. The quaternary structure of ribulose-P2 carboxylase indicates several S to large subunit (L) interactions. Eleven of 22 residues within the loop form the interface with 20 residues from two different L dimers. Eight of the loop residues are involved in hydrogen bonds, salt links, and hydrophobic interactions. To test the hypothesis, whether this loop had a function in the assembly of L and S into the hexadecameric enzyme, 6 amino acids within the loop were modified by site-directed mutagenesis of the pea rbcS-3A gene. All substituted S were imported by isolated chloroplasts from pea with wild type efficiency. Mutants E54-R, H55-A, P59-A, D63-G, D63-L, and Y66-A were assembly-competent, indicating that changes of side chains at these positions are tolerated. Replacement of arginine 53, whose side chain forms H-bonds with L residues Y226 and G261, with glutamate completely abolished assembly into holoenzyme. We suggest that arginine 53 in S is essential for ribulose-P2 carboxylase quaternary structure in higher plants.     

Amino acid-sequence variability at the N-terminal extra piece of mouse immunoglobulin light-chain precursors of the same and different subgroups.

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-63 mouse myeloma L (light) chain were labelled with six radioactive amino acids: [35S]methionine, [4,5-3H]leucine, [3,4-3H]proline, [3-3H]serine, [4,5-3H]isoleucine or [2,3-3H]alanine. Amino acid-sequence analyses showed that over 90% of the total cell-free product was one homogeneous protein, which corresponds to the MOPC-63 L-chain precursor. In this precursor an extra piece, 20 amino acid residues in length, precedes the N-terminus of the mature L chain. The extra piece contains one methionine residue at the N-terminus, six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13, one proline residue at position 16, and one serine residue at position 18. The closely gathered leucine residues, as well as their abundance (30%), suggest that the extra-piece moiety is hydrophobic. In the precursors, the extra piece is coupled to the variable region of the L chain. Partial sequences of precursors of L chains of the same and different subgroups that were labelled with the above six radioactive amino acids indicate that the extra piece is part of the variable region. Thus the precursors of MOPC-63 and MOPC-321 L chains, which are of the same subgroup, have extra pieces of identical size (20 residues), and so far their partial sequences are also identical (see above). On the other hand, in the precursor of MOPC-41 L chain, which is of a different subgroup, the extra piece is 22 residues in length. Further, the sequence of the MOPC-41 extra piece differs in at least ten positions from sequences of the extra pieces of the precursors of MOPC-63 and MOPC-321 L chains.     

Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide-antibody complexes using NMR-derived distance restraints.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.     

An Antioxidant Acidic Polysaccharide from Cuscuta chinensis

An acidic polysaccharide, H2, was isolated from the alkali-extract CHC of seeds of Cuscuta chinensis Lam. with the molecular weight more than 1.0×106. Chemical and spectroscopic studies led to the structure determination as follows: the backbone chain consists of 1,6-linked-β- D Galp, 1,4-linked-β- D Galp, 1,4-linked-β- D GalA and 1,2- or 1,4-linked-β- L Rhap having branching points at position O-3 of some 1,6-linked-β- D Galp residues (one among eight) and O-4 or O-2 of 1,2- or 1,4-linked-β- L Rhap residues to terminal β-D-galactopyranose. The side chains composed of terminal Galp, 1,6-linked-β- D Galp, 1,4-linked β- D Galp and 1,3,6-linked-β- D Galp also linked at position O-3 of 1,6-linked-β- D Galp residues in the backbone chain. β- L -arabinofuranosyl and terminal β- L -rhamnopyranosyl residues existed in the periphery of this polysaccharide linked to O-3 of 1,6-linked-β- D Galp residues in the backbone chain and the side chains. The polysaccharide H2 increased significantly the survival rate of PC12 cells indicating that it had protective effects against H2O2 insult.     

Interactions between cytochrome c2 and photosynthetic reaction center from Rhodobacter sphaeroides: changes in binding affinity and electron transfer rate due to mutation of interfacial hydrophobic residues are strongly correlated

The structure of the complex between cytochrome c(2) (cyt) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides shows contacts between hydrophobic residues Tyr L162, Leu M191, and Val M192 on the RC and the surface of the cyt [Axelrod et al. (2002) J. Mol. Biol. 319, 501-515]. The role of these hydrophobic residues in binding and electron transfer was investigated by replacing them with Ala and other residues. Mutations of the hydrophobic residues generally resulted in relatively small changes in the second-order electron-transfer rate k(2) (Br?nsted coefficient, alpha( )()= 0.15 +/- 0.05) indicating that the transition state for association occurs before short-range hydrophobic contacts are established. Larger changes in k(2), found in some cases, were attributed to a change in the second-order mechanism from a diffusion controlled regime to a rapidly reversible binding regime. The association constant, K(A), of the cyt and the rate of electron transfer from the bound cyt, k(e), were both decreased by mutation. Replacement of Tyr L162, Leu M191, or Val M192 by Ala decreased K(A) and k(e) by factors of 130, 10, 0.6, and 120, 9, 0.6, respectively. The largest changes were obtained by mutation of Tyr L162, showing that this residue plays a key role in both binding and electron transfer. The binding affinity, K(A), and electron-transfer rate, k(e) were strongly correlated, showing that changes of hydrophobic residues affect both binding and electron transfer. This correlation suggests that changes in distance across hydrophobic interprotein contacts have similar effects on both electron tunneling and binding interactions.