Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus.

Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P(+)QB(-) yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB.

High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers.

From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions.

Hydrophobic variants of ganglio-tripod amphiphiles for membrane protein manipulation.

Membrane proteins operate in unique cellular environments. Once removed from their native context for the purification that is required for most types of structural or functional analyses, they are prone to denature if not properly stabilized by membrane mimetics. Detergent micelles have prominently been used to stabilize membrane proteins in aqueous environments as their amphipathic nature allows for shielding of the hydrophobic surfaces of these bio-macromolecules while supporting solubility and monodispersity in water.

Protein influence on charge-asymmetry of the primary donor in photosynthetic bacterial reaction centers containing a heterodimer: effects on photophysical properties and electron transfer.

The substantial electronic distinctions between bacteriochlorophyll (BChl) and its Mg-free analogue bacteriopheophytin (BPh) are exploited in two sets of Rhodobacter capsulatus reaction center (RC) mutants that contain a heterodimeric BChl-BPh primary electron donor (D). The BPh component of the M-heterodimer (Mhd) or L-heterodimer (Lhd) obtains from substituting a Leu for His M200 or for His L173, respectively. Lhd-β and Mhd-β RCs serve as the initial templates in the two mutant sets, where β denotes that the L-side BPh acceptor (HL) has been replaced by a BChl (due to substituting His for Leu M212).

Carbohydrate-containing Triton X-100 analogues for membrane protein solubilization and stabilization.

Membrane protein manipulation is a challenging task owing to limited tertiary and quaternary structural stability once the protein has been removed from a lipid bilayer. Such instability can be overcome by embedding membrane proteins in detergent micelles formed from amphiphiles with carefully tuned properties. This study introduces a class of easy-to-synthesize amphiphiles, which are designated CGT (Chae's Glyco-Triton) detergents.

High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers.

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low.

Glycotripod amphiphiles for solubilization and stabilization of a membrane-protein superassembly: importance of branching in the hydrophilic portion.

Intrinsic membrane proteins must usually be extracted from the native membrane with the aid of synthetic amphiphiles and then stabilized in a soluble form before detailed structural and functional characterization is possible. We describe new amphiphiles with unusual architectures that are useful for extraction and stabilization of photosynthetic protein superassemblies from bacterial membranes. Our results suggest that incorporating branch points in both the hydrophilic and lipophilic portions can lead to favorable amphiphile behavior.