Backbone dynamics and hydrogen exchange of Pseudomonas aeruginosa ferricytochrome c 551

Abstract

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c(551) dynamics based on (15)N R(1), (15)N R(2), and [(1)H]-(15)N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (< S(2)>=0.924+/-0.005) and displays little variation in picosecond-nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (< S(2)>=0.80+/-0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties.

A heme fusion tag for protein affinity purification and quantification

“…The yields we report for AzHm-14 are similar to those of several cyts c overexpressed in E. coli, with the latter having a range of 1-12 mg/L. [27][28][29] The extent of heme attachment to the CXXCH motif has been shown not to depend on the amount of apoprotein having a heme binding motif in the periplasm. 30 To explain this observation, it has been suggested that the availability of free heme transported to the periplasm by an unknown mechanism may determine the yield of protein modified with heme.…”

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c

“…Pa cyt c 551 WT and F7A mutant was prepared as previously reported (33,52). The resonance Raman system (29) and the femtosecond VCS system used in this work have been described in detail elsewhere (26)(27)(28)(29).…”

Cytochrome c552Mutants: Structure and Dynamics at the Active Site Probed by Multidimensional NMR and Vibration Echo Spectroscopy

“…Two-dimensional NMR studies have elucidated the structural and dynamical details of solvated proteins on time scales longer than tens of picoseconds. 13,[25][26][27][28][29][30][31][32][33] Multidimensional IR spectroscopy has improved upon the dynamic range of NMR techniques, revealing biochemical dynamics that occur on the picosecond to femtosecond time scales. [34][35][36][37][38][39][40][41] Vibrational echo spectroscopy is a multidimensional IR technique that is sensitive to the relationship between structure and dynamics in heme proteins.…”