1. Consequences of saturation mutagenesis of the protein ligand to the B-side monomeric bacteriochlorophyll in reaction centers from Rhodobacter capsulatus
Kaitlyn M Faries, Claire E Kohout, Grace Xiyu Wang, Deborah K Hanson, Dewey Holten, Philip D Laible, Christine Kirmaier Photosynth Res. 2019 Sep;141(3):273-290. doi: 10.1007/s11120-019-00626-9. Epub 2019 Mar 11.
In bacterial reaction centers (RCs), photon-induced initial charge separation uses an A-side bacteriochlorophyll (BChl, BA) and bacteriopheophytin (BPh, HA), while the near-mirror image B-side BB and HB cofactors are inactive. Two new sets of Rhodobacter capsulatus RC mutants were designed, both bearing substitution of all amino acids for the native histidine M180 (M-polypeptide residue 180) ligand to the core Mg ion of BB. Residues are identified that largely result in retention of a BChl in the BB site (Asp, Ser, Pro, Gln, Asn, Gly, Cys, Lys, and Thr), ones that largely harbor the Mg-free BPh in the BB site (Leu and Ile), and ones for which isolated RCs are comprised of a substantial mixture of these two RC types (Ala, Glu, Val, Met and, in one set, Arg). No protein was isolated when M180 is Trp, Tyr, Phe, or (in one set) Arg. These findings are corroborated by ground state spectra, pigment extractions, ultrafast transient absorption studies, and the yields of B-side transmembrane charge separation. The changes in coordination chemistries did not reveal an RC with sufficiently precise poising of the redox properties of the BB-site cofactor to result in a high yield of B-side electron transfer to HB. Insights are gleaned into the amino acid properties that support BChl in the BB site and into the widely observed multi-exponential decay of the excited state of the primary electron donor. The results also have direct implications for tuning free energies of the charge-separated intermediates in RCs and mimetic systems.
2. The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit
F J Morgan, S Birken, R E Canfield J Biol Chem. 1975 Jul 10;250(13):5247-58.
The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.
3. Statistical analysis of atomic contacts at RNA-protein interfaces
M Treger, E Westhof J Mol Recognit. 2001 Jul-Aug;14(4):199-214. doi: 10.1002/jmr.534.
Forty-five crystals of complexes between proteins and RNA molecules from the Protein Data Bank have been statistically surveyed for the number of contacts between RNA components (phosphate, ribose and the four bases) and amino acid side chains. Three groups of complexes were defined: the tRNA synthetases; the ribosomal complexes; and a third group containing a variety of complexes. The types of atomic contacts were a priori classified into ionic, neutral H-bond, C-H...O H-bond, or van der Waals interaction. All the contacts were organized into a relational database which allows for statistical analysis. The main conclusions are the following: (i) in all three groups of complexes, the most preferred amino acids (Arg, Asn, Ser, Lys) and the less preferred ones (Ala, Ile, Leu, Val) are the same; Trp and Cys are rarely observed (respectively 15 and 5 amino acids in the ensemble of interfaces); (ii) of the total number of amino acids located at the interfaces 22% are hydrophobic, 40% charged (positive 32%, negative 8%), 30% polar and 8% are Gly; (iii) in ribosomal complexes, phosphate is preferred over ribose, which is preferred over the bases, but there is no significant preference in the other two groups; (iv) there is no significant prevalence of a base type at protein-RNA interfaces, but specifically Arg and Lys display a preference for phosphate over ribose and bases; Pro and Asn prefer bases over ribose and phosphate; Met, Phe and Tyr prefer ribose over phosphate and bases. Further, Ile, Pro, Ser prefer A over the others; Leu prefers C; Asp and Gly prefer G; and Asn prefers U. Considering the contact types, the following conclusions could be drawn: (i) 23% of the contacts are via potential H-bonds (including CH...O H-bonds and ionic interactions), 72% belong to van der Waals interactions and 5% are considered as short contacts; (ii) of all potential H-bonds, 54% are standard, 33% are of the C-H...O type and 13% are ionic; (iii) the Watson-Crick sites of G, O6(G) and principally N2(G) and the hydroxyl group O2' is more often involved in H-bonds than expected; the protein main chain is involved in 32% and the side chains in 68% of the H-bonds; considering the neutral and ionic H-bonds, the following couples are more frequent than expected-base A-Ser, base G-Asp/Glu, base U-Asn. The RNA CH groups interact preferentially with oxygen atoms (62% on the main chain and 19% on the side chains); (iv) the bases are involved in 38% of all H-bonds and more than 26% of the H-bonds have the H donor group on the RNA; (v) the atom O2' is involved in 21% of all H-bonds, a number greater than expected; (vi) amino acids less frequently in direct contact with RNA components interact frequently via their main chain atoms through water molecules with RNA atoms; in contrast, those frequently observed in direct contact, except Ser, use instead their side chain atoms for water bridging interactions.
