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The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides
Published on: November 2, 2021
Structural investigations on novel porin, OmpAb from Acinetobacter baumannii
Jitendra Vashist1, Moganty R Rajeswari
1Department of Biochemistry, All India Institute of Medical Science, New Delhi 110029, India.
Abstract:
Acinetobacter baumannii is an opportunistic pathogen and known to cause nosocomial infections especially in ICUs of hospitals. We have previously reported that the novel outer membrane protein, OmpAb from Acinetobacter baumannii is a transmembrane porin and plays an important role in transport of small molecules, like antibiotics across the membrane. In the present study we report the N-terminal sequence, Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis of OmpAb and structural investigations using UV-Vis absorption, circular dichroism (CD), and fluorescence on OmpAb. SDS-PAGE results suggest that OmpAb is actually a "heat modifiable monomer" and is of 37 kDa at room temperature. Secondary structure of OmpAb is being done for the first time that showed predominantly beta-sheet structure (68%), a feature characteristic of porins. Using N-Bromosuccinimide (NBS) as oxidizing agent, the total number of tryptophans in OmpAb is estimated to be four. The present results indicate that out of the four, two tryptophans seem to be located in the integral part of the membrane, perhaps periplasmic/membrane-bound while the other two tryptophans are exposed to the solvent. We followed the fluorescence emission using conventional 280 nm and selective 305 nm excitation (established by us earlier) to explore the environment of four tryptophans in OmpAb. Emission results using selective excitation of 305 nm revealed local conformational changes of those "tryptophans which are on the surface". On urea denaturation and pH dependent denaturation there is a loss of beta-sheet structure by more than 70%, this is concomitant with the increase in fluorescence intensity and red shift in lambda(max, em). As reflected by CD spectral data, we also found that OmpAb is fairly stable like other porins up to 70 degrees C. As there are no reports on the structural aspects of any outer membrane proteins of Acinetobacter baumannii, results presented here on this novel major porin, OmpAb, will help in understanding the structure-function relationship.
Insights
Acinetobacter baumannii outer membrane protein OmpAb, a porin crucial for antibiotic transport, was structurally investigated. This study reveals its predominantly beta-sheet structure and tryptophan localization, aiding in understanding its function.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Acinetobacter baumannii is a significant nosocomial pathogen.
- The outer membrane protein OmpAb facilitates the transport of small molecules, including antibiotics, across the bacterial membrane.
- Understanding OmpAb's structure is key to combating A. baumannii infections.
Purpose of the Study:
- To characterize the N-terminal sequence and structural properties of OmpAb.
- To investigate the secondary structure, tryptophan environment, and stability of OmpAb.
- To elucidate the structure-function relationship of this novel major porin.
Main Methods:
- Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for N-terminal sequencing and mass analysis.
- UV-Vis absorption, circular dichroism (CD), and fluorescence spectroscopy for structural and conformational studies.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine molecular weight and heat modifiability.
Main Results:
- OmpAb was identified as a heat-modifiable monomer of 37 kDa.
- CD spectroscopy revealed a predominantly beta-sheet structure (68%), characteristic of porins.
- MALDI-TOF MS and N-Bromosuccinimide (NBS) analysis indicated four tryptophan residues, with two likely membrane-bound and two solvent-exposed.
- Fluorescence studies showed local conformational changes upon excitation at 305 nm.
- OmpAb demonstrated stability up to 70°C and significant loss of beta-sheet structure upon urea and pH denaturation.
Conclusions:
- OmpAb possesses a typical porin secondary structure with distinct tryptophan environments.
- The protein exhibits conformational flexibility and stability relevant to its function.
- These findings provide foundational insights into the structure-function relationship of A. baumannii outer membrane proteins.
