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A site-specific double mutant, in which the large aromatic residues M208Tyr and L181Phe in the interior of the photosynthetic reaction center (RC) complex were replaced by smaller threonine residues, showed a dramatic reduction in the number of assembled complexes and was incapable of photosynthetic growth. The cavity created by the smaller side-chains was thought to interfere with the stability and/or assembly of the complex. Phenotypic revertants were recovered in which a spontaneous second-site mutation restored photocompetence in the presence of the original site-specific mutations. In these strains, an Ala→Pro substitution in a neighboring transmembrane helix (at M271) resulted in an increased yield of RC complexes. To test the hypothesis that the original phenotype was due to a cavity, other mutants were constructed that created similar-sized voids at other positions in the membrane-spanning interior. These substitutions caused the same phenotype. Coupling of the above proline substitution to these new cavity mutants also resulted in photocompetent strains that carry increased levels of RC complexes. Therefore, the proline substitution of M271 serves as a global suppressor of the phenotype caused by these internal cavities. The proline substitution slightly increases the thermal stability of the complex at higher temperatures, but the mutant and suppressor strains have about the same stability at the optimal culture temperature, where both are less stable than the wild-type strain. Therefore, the proline substitution may suppress the non-photosynthetic phenotype of cavity mutants by facilitating folding of the nascent polypeptides as they assemble with cofactors to form the transmembranar RC complex. The proline replacement occurs at a pre-existing kink in a transmembrane helix where it can be accommodated without introducing a strain in the structure. The function of proline residues in transmembrane helices might be to promote folding and/or assembly in general.

Periplasmic sensor domains from two methyl-accepting chemotaxis proteins from Geobacter sulfurreducens (encoded by genes GSU0935 and GSU0582) were expressed in Escherichia coli. The sensor domains were isolated, purified, characterized in solution, and their crystal structures were determined. In the crystal, both sensor domains form swapped dimers and show a PAS-type fold. The swapped segment consists of two helices of about 45 residues at the N terminus with the hemes located between the two monomers. In the case of the GSU0582 sensor, the dimer contains a crystallographic 2-fold symmetry and the heme is coordinated by an axial His and a water molecule. In the case of the GSU0935 sensor, the crystals contain a non-crystallographic dimer, and surprisingly, the coordination of the heme in each monomer is different; monomer A heme has His-Met ligation and monomer B heme has His-water ligation as found in the GSU0582 sensor. The structures of these sensor domains are the first structures of PAS domains containing covalently bound heme. Optical absorption, electron paramagnetic resonance and NMR spectroscopy have revealed that the heme groups of both sensor domains are high-spin and low-spin in the oxidized and reduced forms, respectively, and that the spin-state interconversion involves a heme axial ligand replacement. Both sensor domains bind NO in their ferric and ferrous forms but bind CO only in the reduced form. The binding of both NO and CO occurs via an axial ligand exchange process, and is fully reversible. The reduction potentials of the sensor domains differ by 95 mV (-156 mV and -251 mV for sensors GSU0582 and GSU0935, respectively). The swapped dimerization of these sensor domains and redox-linked ligand switch might be related to the mechanism of signal transduction by these chemotaxis proteins.

The packing interactions in crystals of human lambda-type antibody light chain dimers have been reviewed. These homologous proteins are composed of individually specific variable domains, but all have very similar constant domain sequences. The proteins do not emulate each other in their overall crystallization behavior: each attains an individually characteristic space group or unit cell dimensions. However, each of these protein crystals has one unit cell dimension in common, 72.4(+/- 0.2) A. Examination of the protein packing in these crystals reveals that the common cell dimension is a consequence of a packing arrangement of their constant domains, which is conserved in all three crystals. In this striking arrangement, beta-sheets of adjacent constant domains are placed in juxta-position to form an "infinite chain". Although this constant domain packing pattern is rigorously conserved, the variable domain packing arrangements in each of these crystals are different. The conservation of the "infinite" beta-sheet pattern suggests that the constant domain interactions dominate the thermodynamic energy of lattice formation, probably through a combination of specific hydrogen bond formations and by a decrease in the solvent-accessible surface. A single amino acid substitution prohibits this characteristic interneighbor hydrogen bond pattern in the homologous kappa-type light chains. This may explain the observation that very few kappa-type light chains have been crystallized.

A complete human λ Bence Jones protein dimer (Cle) has been isolated and crystallized. Protein Cle was characterized immunochemically and chemically as having a variable region amino acid sequence associated with light chains of the λ chain subgroup, λIII, and a constant region sequence characteristic of “non-Mcg” type λ chains. Bence Jones protein Cle contains two covalently bound intact monomers, each having a molecular weight of ~23,000. Crystals of Bence Jones protein Cle, obtained from ammonium sulfate solutions, diffract to 2.6 A resolution and have the orthorhombic space group P212121 with cell dimensions a = 113.0 A , b = 72.3 A , and c = 48.9 A . The asymmetric unit consists of a dimer with a molecular weight of ~ 46,000.

Available crystallographic data for homologous immunoglobulin constant domains were correlated with measured association constants for these domains. High correlation was found between the association constant and both the buried surface area (number of interdomain contacts) and the number of salt bridges formed in the interaction, whereas no correlation with the number of hydrogen bonds between domains was evident. The total free energy of binding, as determined from the association constant, was related to the number of contacts, hydrogen bonds and salt bridges found in the domain: domain interface by the crystallographic studies. These calculations yielded reasonable average energy terms for each interaction category.

An orthorhombic crystal form ( P 2 1 2 1 2 1 ) of the reaction center from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 has been characterized. The crystals were grown from polyethylene glycol; the unit cell dimensions are a = 142.2 A , b = 139.6 A , and c = 78.7 A ; and they contain one reaction center in each crystallographic asymmetric unit. The crystals diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.

A complete human κ-type Bence—Jones protein (Fin) has been isolated and crystallized. Immunochemical and physicochemical characterization of protein Fin indicates that it is of the κ-chain subgroup, κII, and that it consists of two non-covalently bound intact monomers having a molecular weight of ~23,000 Crystals of Bence—Jones protein Fin obtained from ammonium sulfate solutions have the orthorhombic space group P2 1 2 1 2 1 with cell dimensions a = 132.0 A , b = 93.3 A , and c = 42.3 A . The asymmetric unit consists of a dimer of molecular weight ~46,000.