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[[Image:BetaRoll.jpg|thumb|400px|right|Homology model of a folded Beta Roll domain where the red spheres indicate the bound Ca+2 ions and the purple resides are randomized for molecular recognition (Dooley, Kim, Lu, Tu, and Banta, 2012 Biomacromolecules).]]




'''Evolving the Beta Roll Domain for Regulated Molecular Recognition'''  
'''Evolving the Beta Roll Domain for Regulated Molecular Recognition'''  


Self-assembly is an essential process for all forms of life.  For example, proteins spontaneously fold into well-defined 3-dimensional structures, and cellular organelles form that spatially segregate diverse cellular processes.  As engineers aim to create  new devices and systems at ever decreasing size scales, self-assembly processes become increaseingly attactive techniques.
Molecular recognition is ubiquitous in nature.  Frequently antibodies are used in technology applications where biomolecular recognition is to be employed, but antibodies have several limitations in these applications, including difficulty in easily removing bound targets.  This becomes especially important in the development of biosensors using electrochemical-based signal transduction schemes.
 
We are collaborating with Plamen Atanassov at University of New Mexico, Scott Calabrese-Barton at Michigan State University and Shelley Minteer at Saint Louis University to make improved electrodes for biofuel cells.  In a biofuel cell, redox enzymes are immobilized on electrode surfaces.  Enzymes located at the anode are able to oxidize substrates and the electrons move through an external circuit to create power.  On the cathode, other redox enzymes are able to use the electrons to reduce oxygen to water.  The net result of this process is the generation of electricity from a variety of readily available biofuel sources, with oxygen as the terminal electron acceptor.
 
The architecture of the electrodes is crucial for biofuel cell performance.  The enzymes on the electrodes must be positioned so that electrons can easily move between the enzymatic active site and the electorde surface (Direct Electron Transfer (DET)).  Alternatively, the enzymes can be immoblilized with redox mediators, such as osmium, that facilitate the transport of electrons from the electrodes to the enzymes (Fig. 1).  In this Mediated Electron Transport (MET) configuration, the enzyme and mediators are immobilized in a polymer matrix on the electrode surface.  While this system has been used to demonstrate impressive biofuel cell performances, it is potentially hampered by poor dispersion of the enzyme and mediator within the polymer matrix, and complex manufacturing requirments.


We are using biological self-assembly to improve the biofuel cell electrode construction and performance (Fig. 2).  Instead of combing enzymes and mediators in a polymer matrix, we are creating self-assembling protein-based hydrogels that intrinsically include the redox enzymes and the mediators.  In this configuration the loading of the enzyme and the mediators into the hydrogel can be finely controlled, and the hydrogel assembly process will be well-defined and repeatable.  These new bioelectrocatalytic hydrogels will have the potential to significantly improve biofuel cell performance.
Instead of trying to engineer allosteric control into a molecular recognition molecule, we have started with intrinsically disordered scaffold, the beta roll domain, and we are working to evolve this allosterically regulated scaffold for biomolecular recognition.  The naturally existing beta roll subdomain motif consists of tandem repeats of the sequence GGXGXDXUX, where U is an aliphatic amino acid and X is any amino acid. In the presence of calcium, the disordered peptide reversibly transitions to a beta roll spiral structure of two parallel beta sheet faces, where each beta strand has two solvent exposed variable residues.  


   
We have characterized a native beta roll subdomain with various end-capping groups in order to identify a minimal calcium-responsive beta roll unit. We have immobilized the beta roll on surfaces, and we have developed a FRET-based system to monitor structural perturbations.  We have truncated and concatenated the beta roll and we have discovered beta roll sequences that can reversibly precipitate which is useful for protein purification.  We believe that the beta roll faces are suitable binding surfaces and that calcium-induced structure formation can be used as a mechanism to control the formation of the engineered biomolecular recognition interface.  To test this, we have engineered beta rolls to dimerize in a calcium-dependent manner and used these to create cross-links for protein hydrogels. We have randomized one face of the beta roll unit and we are using directed evolution to identify beta roll peptides with biomolecular recognition capabilities.


'''Related Publications'''
'''Related Publications'''


<biblio>
<biblio>
 
#Paper10 pmid=25226243
#Paper9 pmid=23642248
#Paper8 pmid=23581466
#Paper7 pmid=23173179
#Paper6 pmid=22545587
#Paper5 pmid=21416544
#Paper4 pmid=20438736
#Paper3 pmid=19860484
#Paper3 pmid=19860484
#Paper2 pmid=17376876
#Paper2 pmid=17376876
#Paper1 pmid=17450770
#Paper1 pmid=17450770
</biblio>
</biblio>

Latest revision as of 08:37, 23 October 2014

Banta Lab

Protein and Metabolic Engineering

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Homology model of a folded Beta Roll domain where the red spheres indicate the bound Ca+2 ions and the purple resides are randomized for molecular recognition (Dooley, Kim, Lu, Tu, and Banta, 2012 Biomacromolecules).


Evolving the Beta Roll Domain for Regulated Molecular Recognition

Molecular recognition is ubiquitous in nature. Frequently antibodies are used in technology applications where biomolecular recognition is to be employed, but antibodies have several limitations in these applications, including difficulty in easily removing bound targets. This becomes especially important in the development of biosensors using electrochemical-based signal transduction schemes.

Instead of trying to engineer allosteric control into a molecular recognition molecule, we have started with intrinsically disordered scaffold, the beta roll domain, and we are working to evolve this allosterically regulated scaffold for biomolecular recognition. The naturally existing beta roll subdomain motif consists of tandem repeats of the sequence GGXGXDXUX, where U is an aliphatic amino acid and X is any amino acid. In the presence of calcium, the disordered peptide reversibly transitions to a beta roll spiral structure of two parallel beta sheet faces, where each beta strand has two solvent exposed variable residues.

We have characterized a native beta roll subdomain with various end-capping groups in order to identify a minimal calcium-responsive beta roll unit. We have immobilized the beta roll on surfaces, and we have developed a FRET-based system to monitor structural perturbations. We have truncated and concatenated the beta roll and we have discovered beta roll sequences that can reversibly precipitate which is useful for protein purification. We believe that the beta roll faces are suitable binding surfaces and that calcium-induced structure formation can be used as a mechanism to control the formation of the engineered biomolecular recognition interface. To test this, we have engineered beta rolls to dimerize in a calcium-dependent manner and used these to create cross-links for protein hydrogels. We have randomized one face of the beta roll unit and we are using directed evolution to identify beta roll peptides with biomolecular recognition capabilities.

Related Publications

  1. Dooley K, Bulutoglu B, and Banta S. Doubling the cross-linking interface of a rationally designed beta roll peptide for calcium-dependent proteinaceous hydrogel formation. Biomacromolecules. 2014 Oct 13;15(10):3617-24. DOI:10.1021/bm500870a | PubMed ID:25226243 | HubMed [Paper10]
  2. Banta S, Dooley K, and Shur O. Replacing antibodies: engineering new binding proteins. Annu Rev Biomed Eng. 2013;15:93-113. DOI:10.1146/annurev-bioeng-071812-152412 | PubMed ID:23642248 | HubMed [Paper9]
  3. Shur O, Dooley K, Blenner M, Baltimore M, and Banta S. A designed, phase changing RTX-based peptide for efficient bioseparations. Biotechniques. 2013 Apr;54(4):197-8, 200, 202, 204, 206. DOI:10.2144/000114010 | PubMed ID:23581466 | HubMed [Paper8]
  4. Shur O and Banta S. Rearranging and concatenating a native RTX domain to understand sequence modularity. Protein Eng Des Sel. 2013 Mar;26(3):171-80. DOI:10.1093/protein/gzs092 | PubMed ID:23173179 | HubMed [Paper7]
  5. Dooley K, Kim YH, Lu HD, Tu R, and Banta S. Engineering of an environmentally responsive beta roll peptide for use as a calcium-dependent cross-linking domain for peptide hydrogel formation. Biomacromolecules. 2012 Jun 11;13(6):1758-64. DOI:10.1021/bm3002446 | PubMed ID:22545587 | HubMed [Paper6]
  6. Shur O, Wu J, Cropek DM, and Banta S. Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance. Protein Sci. 2011 May;20(5):925-30. DOI:10.1002/pro.625 | PubMed ID:21416544 | HubMed [Paper5]
  7. Blenner MA, Shur O, Szilvay GR, Cropek DM, and Banta S. Calcium-induced folding of a beta roll motif requires C-terminal entropic stabilization. J Mol Biol. 2010 Jul 9;400(2):244-56. DOI:10.1016/j.jmb.2010.04.056 | PubMed ID:20438736 | HubMed [Paper4]
  8. Szilvay GR, Blenner MA, Shur O, Cropek DM, and Banta S. A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase. Biochemistry. 2009 Dec 1;48(47):11273-82. DOI:10.1021/bi901447j | PubMed ID:19860484 | HubMed [Paper3]
  9. Chockalingam K, Blenner M, and Banta S. Design and application of stimulus-responsive peptide systems. Protein Eng Des Sel. 2007 Apr;20(4):155-61. DOI:10.1093/protein/gzm008 | PubMed ID:17376876 | HubMed [Paper2]
  10. Banta S, Megeed Z, Casali M, Rege K, and Yarmush ML. Engineering protein and peptide building blocks for nanotechnology. J Nanosci Nanotechnol. 2007 Feb;7(2):387-401. DOI:10.1166/jnn.2007.153 | PubMed ID:17450770 | HubMed [Paper1]

All Medline abstracts: PubMed | HubMed

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