Mitsuru Tashiro

Structures of bacterial immunoglobulin-binding domains and their complexes with immunoglobulins

Three-dimensional structures are now available for several immunoglobulin binding domains from bacterial proteins A, G, and L. X-ray diffraction and NMR experiments on complexes of these domains with portions of immunoglobulins have revealed common structural themes used in these interactions. These data expand our understanding of structure/function relationships in these molecular recognition processes and provide the basis for rational design of artificial immunoglobulin-binding molecules.

Phase labeling of C-H and C-C spin-system topologies: application in constant-time PFG-CBCA(CO)NH experiments for discriminating amino acid spin-system types.

SummaryTriple-resonance experiments facilitate the determination of sequence-specific resonance assignments of medium-sized 13C, 15N-enriched proteins. Some triple-resonance experiments can also be used to obtain information about amino acid spin-system topologies by proper delay tuning. The constant-time PFG-CBCA(CO)NH experiment allows discrimination between five different groups of amino acids by tuning (phase labeling) independently the delays for proton-carbon refocusing and carbon-carbon constant-time frequency labeling. The proton-carbon refocusing delay allows discrimination of spin-system topologies based on the number of protons attached to Cα and Cβ atoms (i.e. C-H phase labeling). In addition, tuning of the carbon-carbon constant-time frequency-labeling delay discriminates topologies based on the number of carbons directly coupled to Cα and Cβ atoms (i.e. C-C phase labeling). Classifying the spin systems into these five groups facilitates identification of amino acid types, making both manual and automated analysis of assignments easier. The use of this pair of optimally tuned PFG-CBCA(CO)NH experiments for distinguishing five spin-system topologies is demonstrated for the 124-residue bovine pancreatic ribonuclease A protein.

Classification of amino acid spin systems using PFG HCC(CO)NH-TOCSY with constant-time aliphatic 13C frequency labeling.

SummaryWe have developed a useful strategy for identifying amino acid spin systems and side-chain carbon resonance assignments in small 15N-, 13C-enriched proteins. Multidimensional constant-time pulsed field gradient (PFG) HCC(CO)NH-TOCSY experiments provide side-chain resonance frequency information and establish connectivities between sequential amino acid spin systems. In PFG HCC(CO)NH-TOCSY experiments recorded with a properly tuned constant-time period for frequency labeling of aliphatic 13C resonances, phases of cross peaks provide information that is useful for identifying spin system types. When combined with 13C chemical shift information, these patterns allow identification of the following spin system types: Gly, Ala, Thr, Val, Leu, Ile, Lys, Arg, Pro, long-type (i.e., Gln, Glu and Met), Ser, and AMX-type (i.e., Asp, Asn, Cys, His, Phe, Trp and Tyr).