Dissecting Amelogenin Protein Nanospheres

Bromley, Keith M.; Kiss, Andrew; Lokappa, Sowmya Bekshe; Lakshminarayanan, Rajamani; Fan, Daming; Ndao, Moise; Evans, John Spencer; Moradian‐Oldak, Janet

doi:10.1074/jbc.m111.250928

Cited by 66 publications

(84 citation statements)

References 51 publications

(40 reference statements)

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“…Site-directed mutagenesis was carried out on the DNA template of full-length porcine amelogenin rP172 to generate four mutant proteins, specifically rP172(W25Y,W45Y), rP172[(W25Y,W45Y,W161Y),(F112W)], rP172(W25Y,W161Y), and rP172(W45Y,W161Y), and using Quick Change mutagenesis (Agilent Technologies) as described elsewhere [15,17]. These mutants were designed such that each has only one tryptophan in its entire sequence.…”

Section: Methodsmentioning

confidence: 99%

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Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus

Lokappa

Chandrababu

Moradian‐Oldak

2015

Biochemical and Biophysical Research Communications

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We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residues and fluorescence resonance energy transfer FRET, we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions.

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Section: Methodsmentioning

confidence: 99%

“…Four different amelogenin mutants containing only a single tryptophan (Trp161, Trp112, Trp45 or Trp25) were used for fluorescence analysis [17]. By creating these single-tryptophan amelogenins, which behaved in the same way as the wild type, we were able to identify phospholipid-interacting domains.…”

Section: Introductionmentioning

confidence: 99%

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus

Lokappa

Chandrababu

Moradian‐Oldak

2015

Biochemical and Biophysical Research Communications

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“…Our recent CD analysis has revealed that while oligomer formation accompanies conformational changes in amelogenin, little structural change occurs as a result of nanosphere formation. 54 In their NMR studies, Lu et al . 55 reported a lack of conformational changes in amelogenin nanospheres during gel formation.…”

Section: Amelogenin and Its Targetsmentioning

confidence: 99%

“…We have reported that amelogenin oligomer formation in vitro is mediated by N-terminal protein-protein interactions and assembly, while nanosphere formation is mainly regulated by hydrophobic interactions via the histidine-rich central portion. 54 The question is whether the amelogenin oligomers (R H = 7.5 nm) are the functional entities in enamel formation and that the nanospheres (R H = 14nm) are simply the result of aggregation of oligomers. Particles of 15–20 nm in diameter detected by TEM are the oligomeric structures that were detected in between enamel crystallites.…”

Section: Amelogenin and Its Targetsmentioning

confidence: 99%

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Amelogenin and enamel biomimetics

2015

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Mature tooth enamel is acellular and does not regenerate itself. Developing technologies that rebuild tooth enamel and preserve tooth structure is therefore of great interest. Considering the importance of amelogenin protein in dental enamel formation, its ability to control apatite mineralization in vitro, and its potential to be applied in fabrication of future bio-inspired dental material this review focuses on two major subjects: amelogenin and enamel biomimetics. We review the most recent findings on amelogenin secondary and tertiary structural properties with a focus on its interactions with different targets including other enamel proteins, apatite mineral, and phospholipids. Following a brief overview of enamel hierarchical structure and its mechanical properties we will present the state-of-the-art strategies in the biomimetic reconstruction of human enamel.

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Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles

et al. 2014

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Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172-SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N-terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS-Rosetta modelling confirm that the highly conserved N-terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization.

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Dissecting Amelogenin Protein Nanospheres

Cited by 66 publications

References 51 publications

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus

Amelogenin and enamel biomimetics

Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles

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