Browsing by Subject "Protein cross-linking"
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Item Characterization of the Vibrio cholerae ferrous iron transport system, feo(2015-11-23) Stevenson, Gladys Begoña; Payne, Shelley M.; Browning, Karen S; Davies, Bryan W; Hoffman, David W; Trent, Michael SFeo is the major ferrous iron transport system in prokaryotes and has only been partially characterized, as its assembly and mechanism of transport have not been determined. The feo operon in V. cholerae encodes three proteins, FeoA, FeoB, and FeoC, which are all required for function of the Feo system. FeoA and FeoC are both small cytoplasmic proteins and their function remains unclear. FeoB, thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. However, in this study, a model of the topology of the C-terminal membrane-spanning region of FeoB, based on in vivo labeling experiments, is proposed. Further, through the use of scanning cysteine accessibility mutagenesis, it is determined that the N- and C- termini of FeoB are located in the cytoplasm of V. cholerae. Moreover, epitope-tagged FeoB and FeoC are used to show that these proteins form higher order complexes when cross-linked in vivo in V. cholerae. Further analysis reveals that FeoB simultaneously associates with both FeoA and FeoC to form a large inner membrane complex, an observation that has not been reported previously. It is found that FeoA is required for complex formation, while FeoC is required for wildtype protein levels of FeoB. It is also determined that certain amino acid residues in the GTPase region of FeoB are required for function of the Feo system and for complex formation.Item Development of chromogenic cross-linkers and selective gas-phase dissociation methods to assess protein macromolecular structures by mass spectrometry(2009-12) Gardner, Myles Winston; Brodbelt, Jennifer S.; Marcotte, Edward M.; Shear, Jason B.; Stevens, Scott W.; Stevenson, Keith J.Selective gas-phase dissociation strategies have been developed for the characterization of cross-linked peptides and proteins in quadrupole ion trap mass spectrometers. An infrared chromogenic cross-linker (IRCX) containing a phosphotriester afforded rapid differentiation of cross-linked peptides from unmodified ones in proteolytic digests of cross-linked proteins by selective infrared multiphoton dissociation (IRMPD). Only the cross-linked peptides containing the chromogenic phosphate underwent IRMPD and unmodified peptides were not affected by IR irradiation. IRMPD of IRCX-cross-linked peptides yielded uncross-linked y-ion sequence tags of the constituent peptides due to secondary dissociation of all primary product ions which contained the chromophore, thus allowing successful de novo sequencing of the cross-linked peptides. Peptides cross-linked via a two-step conjugation strategy through the formation of a bis-arylhydrazone (BAH) bond were selectively dissociated by ultraviolet radiation at 355 nm. The BAH-cross-linked peptides could be distinguished from not only unmodified peptides but also dead-end modified peptides based on the selectivity of ultraviolet photodissociation. In a complementary approach, electron transfer dissociation of BAH-cross-linked peptides resulted in preferential cleavage of the hydrazone bond which produced two modified peptides. These modified peptides were subsequently interrogated by CID which allowed for the original site of cross-linking to be pinpointed. IRMPD was implemented in a dual pressure linear ion trap to demonstrate successful photodissociation of peptides having modest absorptivities. Peptides were observed to efficiently dissociation by IR irradiation exclusively in the low pressure cell whereas no dissociation was observed in the high pressure cell due to extensive collisional cooling. IRMPD provided greater sequence coverage of the peptides than CID and yielded product ion mass spectra which were predominantly composed of singly charged product ions which simplified spectral interpretation. IRMPD was further applied for the sequencing of small-interfering RNA. Complete sequence coverage was obtained and the results were compared to CID.