Characterization of Application-Relevant Biosilica-Precipitating Peptides of a Diatom
EMSL Project ID
24798
Abstract
NATURE OF PROPOSAL:
Call for proposal- Science Theme Biological Interactions and Dynamics
Type of proposal- General
Type of Access- Standard
Proprietary status- Nonproprietary
Diatoms have evolved highly detailed mesoporous biosilica cell walls, the frustules, with hierarchically-ordered nanoscale features of interest in fabrication of functional nanomaterials. Frustule formation is directed by silaffins, the biosilica-assembly proteins, and long chain polyamines (LCPAs), which together induce the assembly of ordered biosilica from silicic acid. How silaffins and LCPAs interact in directing biosilica assembly and nanoscale patterning is a central question. A prerequisite and current challenge in addressing this question is obtaining detailed structural characterization of silaffins and their interactions with LCPAs. This proposal describes application of high-precision mass spectrometry for characterization of native silaffins of Thalassiosira pseudonanna and their association with LCPAs with Specific Aims of (1) Characterize the primary structure of silaffins, including all side-chain modifications using high-precision mass spectrometry and (2) Determine the nature of polyamine-silaffin interaction, with focus on identifying specific LCPAs that associate with silaffins and specific sites of noncovalent attachment. We propose to use the conventional "bottom-up" approach to characterize tryptic peptides of silaffins, taking advantage of high-precision MS data to characterize modifications. We also propose to analyze silaffins at the intact protein level using chromatographic fractionation followed by intact protein MS/MS using the complementary approaches of collisionally induced dissociation (CID) and electron capture dissociation (ECD) to identify and locate modifications. To investigate interactions between silaffins and LCPAs, noncovalent interactions will be induced under conditions known to precipitate silica from silicic acid. We propose to use a cross-linking strategy coupled to high-precision MS to characterize the silaffin-polyamine interaction. The cross-linked complexes will be analyzed after trypsin digestion, and also by intact protein MS/MS to identify the sites of polyamine interaction with the silaffins. The findings are expected to contribute to a better understanding of biosilica assembly under ambient conditions of temperature and pressure, conditions central to advancing development of future functional nanomaterials. These techniques would then be available for application to the study of silaffins and polyamines from additional diatom species to learn more about the correlation of frustule conformation with these small molecules.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-07-01
End Date
2010-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members