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Example: Critical Evaluation of a Paper Barthe et al. (2009) Dynamic and Structural Characterization of a Bacterial FHA Protein Reveals a New Autoinhibition Mechanism. Structure, 17: 568 -578 Major Observations & Conclusions: • Odh. I protein is key regulator of the TCA cycle • Unphosphorylated form of Odh. I inhibits the Odh. A protein contains FHA domain highly specific to phosphothreonine § Odhl FHA domain required to bind Odh. A, but Od. HA phosphorylation is unknown § • Phosphorylated form of Odhl is inactive conformational change between phosphorylated and unphosphorylated form § binding of the phosphorylated N-terminal part of the protein to its own FHA domain § new autoinhibition mechanism §
Example: Critical Evaluation of a Paper Major Results: • Identified the Cg. Pkn. A or Cg. Pkn. B phosphorylated site(s) of Odhl by MS as Thr 15 y 1 -y 4 expected mass for the sequence § y 5 -y 10 all masses are less by 98 Da, loss of phosphate group § • Prior identification of Cg. Pkn. G phosphorylation site of Odhl was Thr 14.
Example: Critical Evaluation of a Paper Major Results: • determined NMR structure of unphosphorylated and phosphorylated form of Odhl • Phosphorylation induces folding of N-terminal region short amphipathic helix forms A 20 to S 29 faces Y 11 and P 116 § residues V 12 to F 18 bind to FHA phosphopeptide surface § Interaction between p. T 15 and R 87, R 72 in b 3/b 4 loop and Asn 107 § • Global fold of FHA domain, a b sandwich composed of 11 strands is well conserved b 3/b 4 loop adopt incompatible conformation for phosphopeptide binding in unphosphorylated form §
Example: Critical Evaluation of a Paper Major Results: • N-terminal domain of unphosphorylated form is unstructured and dynamic • phosphorylated form exhibits a decrease in disorder and mobility
Example: Critical Evaluation of a Paper Major Results: • Large chemical shift changes consistent with p. T 15 • Chemical shift changes consistent with ordering of N-terminal region • Chemical shift changes consistent with phosphopeptide binding site
Example: Critical Evaluation of a Paper Major Conclusions Hypothesis: • Phosphorylation at Thr 14 by Cg. Pkn. G could result in a similar inactive Odhl structure • Control of Odh. A activity occurs through phosphorylation of both Odhl and Odh. A • Could not obtain a structure with p. T 14 • Different phosphorylation sites may resulted from different signaling pathways and stimuli Questions: • The importance of the binding of the N-terminal domain to the FHA domain is dependent on the phosphorylation of Odh. A. If the phosphorylation of Odh. A doesn’t occur, then the conformation change and inferred autoinhibition mechanism is not biologically relevant. • Does binding to Odh. A occur through the N-terminal domain or FHA domain? Is N-terminal truncation still active? § Is it the phosphorylation of N-terminal domain or the domain binding to the FHA domain that inactivates Odhl? §
Example: Critical Evaluation of a Paper MW = 609 Questions: • authors claim Thr 15 is phosphorylated §y 5 -y 10 MW = 609? all masses are less by 98 Da, loss of phosphate group NOT TRUE -18 Da § Only evidence is DY 5, if Thr 14 is the phosphorylation site then Y 5 would occur at 609 § Assigned DY 5 peak is weak ~ 3 S/N similar intense peak next to it. Is this 609 peak?
Example: Critical Evaluation of a Paper Questions: • Why are only 1 H-15 N NOEs measured? • Why not measure T 1, T 2 and order parameters (S 2)?
Example: Critical Evaluation of a Paper Questions: • Will the phosphorylated residue or residues next to the phosphorylated site incur the greatest chemical shift change? • Is it T 14 or T 15?