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M Schiffer

Publications and source records attributed to M Schiffer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers.

In Rhodobacter capsulatus, we constructed a quadruple mutant that reversed a structural asymmetry that contributes to the functional asymmetry of the two quinone sites. In the photosynthetically incompetent quadruple mutant RQ, two acidic residues near QB, L212Glu and L213Asp, have been mutated to Ala; conversely, in the QA pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompetent phenotypic revertants (designated RQrev3 and RQrev4) that carry compensatory mutations in both the QA and QB pockets. Near QA, the M246Ala --> Glu mutation remains in both revertants, but M247Asp is replaced by Tyr in RQrev3 and by Ala in RQrev4. The engineered L212Ala and L213Ala substitutions remain in the QB site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the QA and QB sites on electron and proton transfer, we have constructed two additional types of strains. First, "half" revertants were constructed that couple the QB site of the revertants with a wild-type QA site. Second, the QA sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the QB site. We have studied the electron and proton-transfer kinetics on the first and second flashes in reaction centers from these strains by flash-induced absorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246Ala --> Glu + M247Ala --> Tyr mutations near QA. Interestingly, this is not observed when only the M246Ala --> Glu mutation is present in the QA pocket. We suggest that the M247Ala --> Tyr mutation in the QA pocket, or possibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerates the uptake and delivery of protons to the QB anions. The M247Tyr substitution may enable additional pathways for proton transfer that are located near QA.

Alanine↗
PubMed1999

Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.

Amino Acid Sequence↗
PubMed1999

Tertiary structure of human lambda 6 light chains.

AL amyloidosis is a disease process characterized by the pathologic deposition of monoclonal light chains in tissue. To date, only limited information has been obtained on the molecular features that render such light chains amyloidogenic. Although protein products of the major human V kappa and V lambda gene families have been identified in AL deposits, one particular subgroup--lambda 6--has been found to be preferentially associated with this disease. Notably, the variable region of lambda 6 proteins (V lambda 6) has distinctive primary structural features including the presence in the third framework region (FR3) of two additional amino acid residues that distinguish members of this subgroup from other types of light chains. However, the structural consequences of these alterations have not been elucidated. To determine if lambda 6 proteins possess unique tertiary structural features, as compared to light chains of other V lambda subgroups, we have obtained x-ray diffraction data on crystals prepared from two recombinant V lambda 6 molecules. These components, isolated from a bacterial expression system, were generated from lambda 6-related cDNAs cloned from bone marrow-derived plasma cells from a patient (Wil) who had documented AL amyloidosis and another (Jto) with multiple myeloma and tubular cast nephropathy, but no evident fibrillar deposits. The x-ray crystallographic analyses revealed that the two-residue insertion located between positions 68 and 69 (not between 66 and 67 as previously surmised) extended an existing loop region that effectively increased the surface area adjacent to the first complementarity determining region (CDR1). Further, an unusual interaction between the Arg 25 and Phe 2 residues commonly found in lambda 6 molecules was noted. However, the structures of V lambda 6 Wil and Jto also differed from each other, as evidenced by the presence in the latter of certain ionic and hydrophobic interactions that we posit increased protein stability and thus prevented amyloid formation.

Amino Acid Sequence↗
PubMed1998

A domain flip as a result of a single amino-acid substitution.

BACKGROUND: The self-assembly properties of beta domains are important features of diverse classes of proteins that include cell-adhesion molecules, surface receptors and the immunoglobulin superfamily. Immunoglobulin light-chain variable domains are well suited to the study of structural factors that determine dimerization, including how residues at the interface influence the preferred dimer arrangement. RESULTS: Single-site mutants of light-chain variable domain Len, designated LenQ38E and LenK30T, formed 'flipped' dimers in which one domain was rotated by about 180 degrees compared with the native protein. The dimer in the native protein is similar to that found between variable domains in Fab immunoglobulin fragments. When compared to the native dimer, more surface area is buried, and more hydrogen bonds and salt bridges are formed between the monomers in the flipped conformation. CONCLUSIONS: Immunoglobulin light-chain variable domains can form a minimum of two distinct quaternary structures. Single-site mutations resulting from changes of one base, such as the exchange of Gln38 to Glu or Lys30 to Thr, change the 'conventional' dimer of protein Len to a flipped arrangement. Native Len is not found in the flipped-domain dimer conformation because it would have excess positive electrostatic potential at the dimer interface that is not compensated by other forces. Excess negative or positive electrostatic potential at the dimer interface can have a determining effect on the mode of dimerization.

Crystallography, X-Ray↗
PubMed1998

Flash-induced changes in buffering capacity of reaction centers from photosynthetic bacteria reveal complex interaction between quinone pockets.

A novel method was applied to determine light-induced protonation in reaction centers from photosynthetic purple bacteria. Changes in buffering capacities upon flash excitation were detected in (0.03% Triton X-100) detergent solution of reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus wild type and mutant strains with empty or occupied secondary quinone (QB) binding sites in the presence of an external electron donor. The light-induced differences in buffering capacities between pH 4 and 11 were analyzed in terms of protonatable residues. Due to its differential nature, this method is more sensitive to the position and shift of pKa of the individual groups than the direct method based on proton uptake measurements. Out of the four different ionizable residues which were used to fit the curves, the two groups with apparent (dark) pKa values between 8.4-8.8 and 9.5-10.0 (depending on the species and conditions) disappeared when the native ubiquinone10 was replaced by menadione at the primary quinone (QA) binding site of Rb. sphaeroides or when the key protonatable residues (L212Glu and L213Asp) were replaced by non-protonatable alanines in the QB binding site of the AA+M43D mutant from Rb. capsulatus. The experimentally observed acidic and neutral residues remained unchanged. These results obtained from modifications in both quinone sites reveal the origin of the alkaline pH groups: they reflect the interaction of QA- and the cluster of ionizable residues around L212Glu in the QB binding pocket. The involvement of two residues with close pKa values reflects the complex titration of the cluster. The interaction between the quinone pockets is best described qualitatively as a network of ionizable residues extending from the QA site to the QB site.

Journal Article↗
PubMed1998

In bacterial reaction centers, a key residue suppresses mutational blockage of two different proton transfer steps.

In reaction centers of Rhodobacter (Rb.) capsulatus, the M43Asn --> Asp substitution is capable of restoring rapid rates for delivery of the second proton to QB in a mutant that lacks L212Glu. Flash-induced absorbance spectroscopy was used to show a nearly native rate for transfer of the second proton to QB (approximately 700 s-1) in the L212Gln+M43Asp double-mutant reaction center; this rate was shown to decrease more than 1000-fold in the photoincompetent L212Glu --> Gln mutant [Miksovska, J., Kálmán, L., Maróti, P., Schiffer, M., Sebban, P., and Hanson, D.K. (1997) Biochemistry 36, 12216-12226]. In Rb. sphaeroides, the equivalent M44Asn --> Asp mutation was reported to restore the rate of transfer of the first proton to a wild-type level when it is added to the L213Asp --> Asn photoincompetent mutant [Rongey, S.H., Paddock, M.L., Feher, G., and Okamura, M.Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1325-1329]. It is remarkable that the same second-site mutation can compensate for both of these mutations which severely impair reaction center function by blocking two different proton-transfer reactions. We suggest that residue M43Asp is situated in a key position which can link pathways for delivery of both the first and second protons (involving structured water molecules) to QB.

Cytochrome c Group↗
PubMed1998

Protein modifications affecting triplet energy transfer in bacterial photosynthetic reaction centers.

The efficiency of triplet energy transfer from the special pair (P) to the carotenoid (C) in photosynthetic reaction centers (RCs) from a large family of mutant strains has been investigated. The mutants carry substitutions at positions L181 and/or M208 near chlorophyll-based cofactors on the inactive and active sides of the complex, respectively. Light-modulated electron paramagnetic resonance at 10 K, where triplet energy transfer is thermally prohibited, reveals that the mutations do not perturb the electronic distribution of P. At temperatures > or = 70 K, we observe reduced signals from the carotenoid in most of the RCs with L181 substitutions. In particular, triplet transfer efficiency is reduced in all RCs in which a lysine at L181 donates a sixth ligand to the monomeric bacteriochlorophyll B(B). Replacement of the native Tyr at M208 on the active side of the complex with several polar residues increased transfer efficiency. The difference in the efficiencies of transfer in the RCs demonstrates the ability of the protein environment to influence the electronic overlap of the chromophores and thus the thermal barrier for triplet energy transfer.

Amino Acid Substitution↗
PubMed1998

Reengineering immunoglobulin domain interactions by introduction of charged residues.

The formation of the antibody variable domain binding unit (Fv) is the net result of three competing assembly reactions. The affinities of concurrent homologous interactions of heavy and light chain variable domains limits the heterologous interaction leading to productive formation of the Fv. To address the possible role of light chain dimerization in this phenomenon, the Gln38 residue at the dimer interface of an immunoglobulin light chain variable domain (VL) was replaced by charged amino acids. The effects of these mutations on VL homodimer formation were monitored by small-zone size exclusion HPLC and the affinities of interaction were determined by computer simulation. Reduced VL homodimerization was observed in three of the four mutants, Q38R, Q38D and Q38K. The association constants for the Q38R and Q38D homodimers were 1.2 x 10(4) and 3.2 x 10(3) M(-1), respectively. This corresponded to a 20-75-fold reduction in the homodimer association constant relative to the wild-type VL, which had an association constant of 2.4 x 10(5) M(-1). Surprisingly, the fourth charge mutant, Q38E, had a higher association constant than the wild-type VL. The potential for charged residues to facilitate heterodimeric assembly of immunoglobulin domains was also tested. Heterodimerization was observed between the Q38D and Q38R V(L)s, but with an association constant of 4.7 x 10(4) M(-1), approximately fivefold lower than that obtained for homodimerization of the native V(L). In addition, replacement of the neutral, solvent-accessible Gln38 residue with either Asp or Arg was found to be significantly destabilizing. These results suggest that charged residues could be introduced at immunoglobulin domain interfaces to guide heterodimer formation and to minimize unfavorable competing homologous associations. Nonetheless, these apparently simple modifications may also result in unintended consequences that are likely to depend upon structural features of particular variable domains.

Chromatography, Gel↗
PubMed1997

In bacterial reaction centers rapid delivery of the second proton to QB can be achieved in the absence of L212Glu.

In the reaction center (RC) of Rhodobacter capsulatus, residue L212Glu is a component of the pathway for proton transfer to the reduced secondary quinone, QB. We isolated phenotypic revertants of the photosynthetically incompetent (PS-) L212Glu-->Gln mutant; all of them retain the L212Glu-->Gln substitution and carry a second-site mutation: L227Leu-->Phe, L228Gly-->Asp, L231Arg-->Cys, or M231Arg-->Cys. We also characterized the L212Ala strain, which is a phenotypic revertant of the PS- L212Glu-L213Asp-->Ala-Ala mutant. The activities of the RCs of these strains--all of which lack L212Glu--were studied by flash-induced absorption spectroscopy. At pH 7.5, the rate of second electron transfer in the L212Q mutant is comparable to the wild-type rate. However, this mutant shows a marked decrease in the rate of cytochrome oxidation under strong continuous illumination and a very slow phase (0.66 s-1) of the proton transfer kinetics following the second flash, indicating that transfer of the second proton to QB is slowed more than 1000-fold. The levels of recovery of the functional capabilities in the revertant RCs vary widely; their rates of cytochrome oxidation were intermediate between those of the wild-type and the L212Q mutant. The kinetics of proton transfer following the second flash show a significant recovery in the L212Q + M231C and L212A RCs (330-540 s-1), but the L212Q + L227F RCs recover this function only partially. Compensation for the lack of L212Glu in revertant RCs is discussed in terms of (i) conformational changes that could allow water molecules to approach closer to QB and/or (ii) the increase in the negative electrostatic environment and the resultant rise in the free energy level of QB- that is induced by the mutations. The stoichiometries of H+/QB- proton uptake below pH 7.5 in the L212Q mutant, the L212Q + M231C revertant, and the wild-type strains are essentially equivalent, suggesting that L212Glu is protonated at neutral pH in wild-type RCs. This is also supported by the P+QB- charge recombination data. Comparison of H+/QB- proton uptake data with those obtained previously for the stoichiometries of H+/QA- proton uptake [Miksovska, J., Maróti, P., Tandori, J., Schiffer, M., Hanson, D. K., Sebban, P. (1996) Biochemistry 35, 15411-15417] suggests that L212Glu is the key to the electrostatic and perhaps structural interaction between the two quinone sites.

Cytochrome c Group↗
PubMed1997

A native electrostatic environment near Q(B) is not sufficient to ensure rapid proton delivery in photosynthetic reaction centers.

Flash-induced absorption spectroscopy has been used to characterize Rhodobacter capsulatus reaction centers mutated in the secondary quinone acceptor site (Q(B). We compared the wild-type, the L212Glu-L213Asp --> Ala-Ala photosynthetically incompetent double mutant (DM), and two photocompetent revertants, the DM+L217Arg --> Cys and the DM+M5Asn- --> Asp strains. The electrostatic environment for Q(B)- is different in the two revertant strains. Only the L217Arg --> Cys mutation nearly restores the native electrostatic environment of Q(B)-. However, the level of recovery of the reaction center function, measured by the rates of second electron transfer and cytochrome c turnover, is quite incomplete in both strains. This shows that a wild-type-like electrostatic environment of Q(B)- cannot ensure on its own, rapid and efficient proton transfer to Q(B)-.

Cytochrome c Group↗
PubMed1997

Variable domain structure of kappaIV human light chain Len: high homology to the murine light chain McPC603.

Antibody light chains of the kappa subgroup are the predominant light chain component in human immune responses and are used almost exclusively in the antibody repertoire of mice. Human kappa light chains comprise four subgroups. To date, all crystallographic studies of human kappa light chains were carried out on proteins of the kappaI subgroup. The light chain produced by multiple myeloma patient Len. was of the kappaIV subgroup, it differed by only one residue from the germ-line gene encoded protein. The variable domain fragment of the light chain was crystallized from ammonium sulfate in space group C222(1). The crystal structure was determined by molecular replacement and refined at 1.95 A resolution to an R-factor of 0.15. Protein Len has six additional residues in its CDR1 segment compared to the kappaI proteins previously characterized. The kappaIV variable domain, Len, differs in only 23 of 113 residues from murine kappa light chain McPC603. The RMS deviation upon superimposing their alpha-carbons was 0.69 A. The CDR1 segment of the human and murine variable domains have the same length and conformation although their amino acid sequences differ in 5 out of 17 residues. Structural features were identified that could account for the significantly higher stability of the human kappaIV protein relative to its murine counterpart. This human kappaIV light chain structure is the closest human homolog to a murine light chain and can be expected to facilitate detailed structural comparisons necessary for effective humanization of murine antibodies.

Amino Acid Sequence↗
PubMed1996

Distant electrostatic interactions modulate the free energy level of QA- in the photosynthetic reaction center.

In the reaction centers from the purple photosynthetic bacterium Rhodobacter capsulatus, we have determined that residue L212Glu, situated near the secondary quinone acceptor QB, modulates the free energy level of the reduced primary quinone molecule QA- at high pH. Even though the distance between L212Glu and QA is 17 A, our results indicate an apparent interaction energy between them of 30 +/- 18 meV. This interaction was measured by quantitating the stoichiometry of partial proton uptake upon formation of QA- as a function of pH in four mutant strains which lack L212Glu, in comparison with the wild type. These strains are the photosynthetically incompetent site-specific mutants L212Glu -->Gln and L212Glu-L213Asp-->Ala-Ala and the photocompetent strains L212Glu-->Ala and L212Ala-L213Ala-M43Asn-->Ala-Ala-Asp. Below pH 7.5, the stoichiometry of proton uptake from all strains is nearly superimposable with that of the wild type. However, at variance with the wild type, reaction centers from all strains that lack L212Glu fail to take up protons above pH 9. The lack of a change in the free energy level of QA- at high pH in the L212Glu-modified strains is confirmed by the determination of the pH dependence of the rate (kAP) of P+QA- charge recombination in the reaction centers where the native QA is replaced by quinones having low redox potentials. Contrary to the wild-type reaction centers where kAP increases at high pH, almost no pH dependence could be detected in the strains that lack L212Glu. Our data show that the ionization state of L212Glu, either on its own or via interactions with closely associated ionizable groups, is mainly involved in the proton uptake at high pH by reaction centers in the PQA- state. This suggests that the formation of the QA- semiquinone state induces shifts in pKas of residues in the QB proteic environment. This long-distance influence of ionization states is a mechanism which would facilitate electron transfer from QA to QB on the first and second flashes. The functional communication between the two quinone protein pockets may involve the iron-ligand complex which spans the distance between them.

Benzoquinones↗
PubMed1996

Pitfalls of molecular replacement: the structure determination of an immunoglobulin light-chain dimer.

The structure of protein Cle, a human light-chain dimer from the lambdaIII subgroup, was determined using 2.6 A data; the R value is 18.4%. The structure was solved, after a false start, by molecular replacement with the lambdaII/V Mcg protein as a search structure. When the refinement did not proceed beyond an R value of 27%, it was discovered that while the constant domains were in their correct positions in the unit cell, the incorrect variable domains were used for defining the molecule. The correct solution required a rotation of 180 degrees around the local twofold axis that relates the two constant domains of the dimer. The correct variable domain positions overlap about 70% of the same volume as the incorrect ones of a symmetry-related molecule. The refinement distorted the geometries of the domains. Though the constant domains were in their correct positions, the r.m.s. (root-mean-square) deviation of the Calpha atom position was 1.2 A when the two constant domains were compared. For the correct structure, this value is 0.5 A. The phi and psi angles, the r.m.s. chiral value and the free R value, even when calculated a posteriori, were good indicators of the correctness of the structure. The quaternary structure of the Cle molecule is similar to that in Mcg (crystallized from ammonium sulfate); the elbow bend is 115 degrees. However, the arrangement of the variable domains differs from that observed in other variable domain dimers. The variable domains of Cle are 0.7 A closer than in Mcg or variable dimer Rei. The hydrogen bonding at the interface of the two domains is novel. Residues Tyr36 from both monomers form a hydrogen bond that is part of a network with the Gln89 residues from both monomers. For the first time hydrogen bonds were observed between the main-chain peptide N and O atoms of the complementarity-determining region CDR2 and CDR3 segments of both monomers.

Journal Article↗
PubMed1996

Three quaternary structures for a single protein.

The structure of a multisubunit protein (immunoglobulin light chain) was solved in three crystal forms, differing only in the solvent of crystallization. The three structures were obtained at high ionic strength and low pH, high ionic strength and high pH, and low ionic strength and neutral pH. The three resulting "snapshots" of possible structures show that their variable-domain interactions differ, reflecting their stabilities under specific solvent conditions. In the three crystal forms, the variable domains had different rotational and translational relationships, whereas no alteration of the constant domains was found. The critical residues involved in the observed effect of the solvent are tryptophans and histidines located between the two variable domains in the dimeric structure. Tryptophan residues are commonly found in interfaces between proteins and their subunits, and histidines have been implicated in pH-dependent conformation changes. The quaternary structure observed for a multisubunit protein or protein complex in a crystal may be influenced by the interactions of the constituents within the molecule or complex and/or by crystal packing interactions. The comparison of buried surface areas and hydrogen bonds between the domains forming the molecule and between the molecules forming the crystals suggest that, for this system, the interactions within the molecule are most likely the determining factors.

Ammonium Sulfate↗
PubMed1996

Novel unconventional binding site in the variable region of immunoglobulins.

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

Affinity Labels↗