protein engineered nanomaterialsprotein engineered nanomaterials dr. jin kim montclare complex...
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![Page 1: Protein Engineered NanomaterialsProtein Engineered Nanomaterials Dr. Jin Kim Montclare Complex functions in organisms are achieved through proteins Building Blocks of Nature: Proteins](https://reader036.vdocuments.site/reader036/viewer/2022070802/5f02bec37e708231d405ccfd/html5/thumbnails/1.jpg)
Protein Engineered Nanomaterials
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![Page 3: Protein Engineered NanomaterialsProtein Engineered Nanomaterials Dr. Jin Kim Montclare Complex functions in organisms are achieved through proteins Building Blocks of Nature: Proteins](https://reader036.vdocuments.site/reader036/viewer/2022070802/5f02bec37e708231d405ccfd/html5/thumbnails/3.jpg)
Dr. Jin Kim MontclareAssociate ProfessorDirector, Convergence for Innovation & Entrepreneurship (CIE) InstituteNew York University
Dr. Priya KatyalPostdoctoral AssociateNew York University
Lindsay K. HillMD/PhD candidate New York UniversitySUNY Downstate Medical Center
Presenters
![Page 4: Protein Engineered NanomaterialsProtein Engineered Nanomaterials Dr. Jin Kim Montclare Complex functions in organisms are achieved through proteins Building Blocks of Nature: Proteins](https://reader036.vdocuments.site/reader036/viewer/2022070802/5f02bec37e708231d405ccfd/html5/thumbnails/4.jpg)
• Proteins as building blocks
• Engineered protein fibers
• Engineered protein nanoparticles
Protein Engineered Nanomaterials
![Page 5: Protein Engineered NanomaterialsProtein Engineered Nanomaterials Dr. Jin Kim Montclare Complex functions in organisms are achieved through proteins Building Blocks of Nature: Proteins](https://reader036.vdocuments.site/reader036/viewer/2022070802/5f02bec37e708231d405ccfd/html5/thumbnails/5.jpg)
• Proteins as building blocks
• Engineered protein fibers
• Engineered protein nanoparticles
Protein Engineered Nanomaterials
Dr. Jin Kim Montclare
![Page 6: Protein Engineered NanomaterialsProtein Engineered Nanomaterials Dr. Jin Kim Montclare Complex functions in organisms are achieved through proteins Building Blocks of Nature: Proteins](https://reader036.vdocuments.site/reader036/viewer/2022070802/5f02bec37e708231d405ccfd/html5/thumbnails/6.jpg)
Complex functions in organisms are achieved through proteins
Building Blocks of Nature: Proteins
Dr. Jin Kim Montclare
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Novel functions achieved through “artificial” proteins
20 amino acid alphabet with defined order and number of secondary structural elements
Non-canonical amino acids
Building Blocks of Nature: Proteins
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Program Bacteria: Living Factories for Artificial Proteins
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• Mono-dispersity
Proteins
(PLGAs)
Synthetic Polymer
(PMMA)
I. Chen and B. Gao Anal. Chem., 1997, 69, 4399
• Well defined secondary structures
[(AG)xEG]n
Krejchi et.al. Science, 1994, 265, 1427
Chain length, sequence and stereochemistry
Advantages of Biopolymer Synthesis
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Nanofibers NanoparticlesLipoproteoplexes
…for treating various disorders:• Osteoarthritis• Breast cancer• Diabetic wound healing• Diagnostic and imaging
Rabbani, P., et al. Biomaterials 2017; 132: 1-15.Hume, J, et al. Biomacromolecules 2014; 15: 3503-3510.Gunasekar S.K. et al., Biochemistry, 2009, 48, 8559-8567Liu, C.F., Chen, R. et al. Biomacromolecules 2017; 18: 2688-2698.
Hydrogels
Montclare Lab: Constructing smart biomaterials
More, H.T., et al. Biomaterials 2014; 35: 7188-7193.Haghpanah, J. et al., Mol. BioSyst., 2010; 6:1662-1667Dai, M, et al. Biomacromolecules 2011; 12: 4240-4246.Haghpanah, J. et al., ChemBioChem, 2009; 10:2733-2735Dai, M., Frezzo, J.A. et al. J. Nanomed Nanotechnol. 2016; 7:356.
Mesofibers
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• Proteins as building blocks
• Engineered protein fibers
• Engineered protein nanoparticles
Objectives
Lindsay K. Hill
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Cartilage Oligomeric Matrix Protein
Smith R.K.W. et al. Matrix Biology.1997; 16, 255-271Posey, K.L. et al. Current Drug Targets. 2008; 9, 869-877Geng H, et al. Arthritis Res Ther. 2008; 10(6): R134.
•Four-domain non-collagenous extracellular matrix protein•Found in cartilage, ligaments, and tendons•N-terminal maintains a coiled-coil structure
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Cartilage Oligomeric Matrix Protein, Coiled-coil Domain (COMPcc)
•COMP’s N-terminal domain maintains a coiled-coil structure
S. Özbek et al. The EMBO Journal 2002, 21, 5960-5968Malashkevich, V.N. et al. Science. 1996; 274:761-765Gunasekar SK, et al. Adv. Funct. Mater. 22, 2154–2159 (2012).
MRGSHHHHHHGSGDL APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNCOMPcc:
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•Green = protein COMPcc•Grey = small molecule cargo
Cartilage Oligomeric Matrix Protein, Coiled-coil Domain (COMPcc)
S. Özbek et al. The EMBO Journal 2002, 21, 5960-5968Malashkevich, V.N. et al. Science. 1996; 274:761-765Gunasekar SK, et al. Adv. Funct. Mater. 22, 2154–2159 (2012).
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Gunasekar SK, et al. Adv. Funct. Mater.2012; 22, 2154–2159.
COMPcc10-15 nm
COMPcc Fibers
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Gunasekar SK, et al. Adv. Funct. Mater.2012; 22, 2154–2159.
COMPcc Single-residue Mutants
MRGSHHHHHHGSGDL APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNCOMPcc:
MRGSHHHHHHGSGDL APQMLRE LQEANAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNT40A:
MRGSHHHHHHGSGDL APQMLRE LQETNAA AQDVREL LRQQVKE ITFLKNT VMESDAS GKLNL44A:
COMPcc T40A L44A
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Gunasekar SK, et al. Adv. Funct. Mater. 2012; 22, 2154–2159.Rowinski-Zyrek M, et al. New J. Chem. 2013: 37, 58-70.
COMPcc Single-residue Mutants
MRGSHHHHHHGSGDL APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNCOMPcc:
MRGSHHHHHHGSGDL APQMLRE LQEANAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNT40A:
MRGSHHHHHHGSGDL APQMLRE LQETNAA AQDVREL LRQQVKE ITFLKNT VMESDAS GKLNL44A:
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Gunasekar SK, et al. Adv. Funct. Mater.2012; 22, 2154–2159.
COMPcc Single-residue Mutants
COMPcc
T40A
L44A
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Circular Dichroism Spectroscopy (CD): Structural Assessment
Right and Left-HandedCircularly Polarized Light
PhotonBeam Optically Active
Sample (e.g. protein)
CD Signal
©2006 NMSU Board of Regents..
- Structured- Reduced structure- Loss of structure
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Gunasekar SK, et al. Adv. Funct. Mater.2012; 22, 2154–2159.
COMPcc Single-residue Mutants
MRGSHHHHHHGSGDL APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNCOMPcc:
MRGSHHHHHHGSGDL APQMLRE LQEANAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNT40A:
MRGSHHHHHHGSGDL APQMLRE LQETNAA AQDVREL LRQQVKE ITFLKNT VMESDAS GKLNL44A:
COMPcc: T40A: L44A:
+Zn(II) +Zn(II)+Zn(II)
+Ni(II)+Ni(II)
+Ni(II)
COMpccT40A
L44A
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Hume, J, et al. Biomacromolecules 2014; 15: 3503-3510.Gunasekar SK, et al. Adv. Funct. Mater. 2012; 22, 2154–2159
COMPcc Variants
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COMPcc Variants
Hume, J, et al. Biomacromolecules 2014; 15: 3503-3510.Gunasekar SK, et al. Adv. Funct. Mater. 2012; 22, 2154–2159
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Domain swapped COMPcc: Q protein
MRGSHHHHHHGSIEGR VKE ITFLKNT APQMLRE LQETNAA LQDVREL LRQQSKL Q:
Hume, J, et al. Biomacromolecules 2014; 15: 3503-3510.Gunasekar SK, et al. Adv. Funct. Mater. 2012; 22, 2154–2159
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Surface charge
COMPcc Q
Domain swapped COMPcc: Q protein
Hume, J, et al. Biomacromolecules 2014; 15: 3503-3510.Gunasekar SK, et al. Adv. Funct. Mater. 2012; 22, 2154–2159
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Surface charge
COMPcc Q
Domain swapped COMPcc: Q protein
Q nanofibersHume, J, et al. Biomacromolecules 2014; 15: 3503-3510.Gunasekar SK, et al. Adv. Funct. Mater. 2012; 22, 2154–2159
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Domain swapped COMPcc: Q protein
Hume, J, et al. Biomacromolecules 2014; 15: 3503-3510.Gunasekar SK, et al. Adv. Funct. Mater. 2012; 22, 2154–2159
Surface charge
COMPcc Q
COMPcc10-15 d.nm
Q20-560 d.nm
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Rahmni, A.H. et al., Biomed. Res. Int. , 2014; 761608
•Active ingredient in turmeric•Has anti-cancer, anti-inflammatory, and anti-bacterial•Absorbs at 420 nm•Fluoresces at 530 nm when protein-bound
Curcumin: Derived from Turmeric
Domain swapped COMPcc: Q proteinSmall Drug Binding
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Hume, J, et al. Biomacromolecules 2014; 15: 3503-3510.
Q20-560 d.nm +
curcumin
Q16.0 +/- 5.6 d.µm
Nano-scale Fibers(Nanofibers)
Micron-scale Fibers(Mesofibers)
Domain swapped COMPcc: Q proteinSmall Drug Binding
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More HT, et al. Biomacromolecules 2015; 16: 1210-1217.
Leucine Trifluoroleucine•Naturally-occurring amino acid •Non-natural/ non-canonical amino acid
•Includes three fluorine atomsFluorine:•Improves protein stability against heat and chemicals•Fluorine MRI (limited fluorine in body signal is specific to the construct)
Fluorinated Q protein
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Leucine Trifluoroleucine•Naturally-occurring amino acid •Non-natural/ non-canonical amino acid
•Includes three fluorine atomsFluorine:•Improves protein stability against heat and chemicals•Fluorine MRI (limited fluorine in body signal is specific to the construct)
Fluorinated Q protein
More HT, et al. Biomacromolecules 2015; 16: 1210-1217.Flögel U, et al. Circulation 2008; 118: 140-148.
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Metal Binding by Q+TFL
+ Zinc
+ Nickel
•Zinc-stabilized fibers
•Nickel disrupts fiber formation and yields aggregation
•TFL-incorporated Q fibers have increased thermostability and chemical stability
Fluorinated Q protein
More HT, et al. Biomacromolecules 2015; 16: 1210-1217.
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• Proteins as building blocks
• Engineered protein fibers
Protein Engineered Nanomaterials
Any Questions?
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• Proteins as building blocks
• Engineered protein fibers
• Engineered protein nanoparticles
Objectives
Dr. Priya Katyal
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More, H.T., et al. Biomaterials 2014; 35: 7188-7193.Gunasekar SK, et al. Adv. Funct. Mater. 22, 2154–2159 (2012).
COMPcc Supercharged Protein (CSP)
Surface charge
COMPcc
MRGSHHHHHHGSGDL APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNCOMPcc:
MRGSHHHHHHGSGRL RPQMLRE LQRTNAA LRDVREL LRQQVKE ITRLKNT VRRSRAS GKLNCSP
Dr. Priya Katyal
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Nucleic acid (siRNA)
Nucleic acid Binding
More, H.T., et al. Biomaterials 2014; 35: 7188-7193.Liu, C.F., Chen, R. et al. Biomacromolecules 2017; 18: 2688-2698.
++
++++++++
++
+++++
+++
- - - - - - - -- - - - - - - -
COMPcc Supercharged Protein (CSP)
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Nucleic acid (siRNA)
Proteoplex particle
Nucleic acid Binding
More, H.T., et al. Biomaterials 2014; 35: 7188-7193.Liu, C.F., Chen, R. et al. Biomacromolecules 2017; 18: 2688-2698.
++
++++++++
++
+++++
+++
- - - - - - - -- - - - - - - -
COMPcc Supercharged Protein (CSP)
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Nucleic acid (siRNA)
Proteoplex particle
Liposomes
CSP-Lipid assembliesNucleic acid Binding
More, H.T., et al. Biomaterials 2014; 35: 7188-7193.Liu, C.F., Chen, R. et al. Biomacromolecules 2017; 18: 2688-2698.
++
++++++++
++
+++++
+++
- - - - - - - -- - - - - - - -
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Nucleic acid (siRNA)
Proteoplex particle
Liposomes
Lipoproteoplex(Lipid-protein complex)
CSP-Lipid assembliesNucleic acid Binding
More, H.T., et al. Biomaterials 2014; 35: 7188-7193.Liu, C.F., Chen, R. et al. Biomacromolecules 2017; 18: 2688-2698.
++
++++++++
++
+++++
+++
- - - - - - - -- - - - - - - -
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Nucleic acid (siRNA)
Proteoplex particle
Liposomes
Lipoproteoplex(Lipid-protein complex)
CSP-Lipid assembliesNucleic acid Binding
More, H.T., et al. Biomaterials 2014; 35: 7188-7193.Liu, C.F., Chen, R. et al. Biomacromolecules 2017; 18: 2688-2698.
++
++++++++
++
+++++
+++
- - - - - - - -- - - - - - - -
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CSP-Lipid assembliesDiabetic Wound Healing
Rabbani, P., et al. Biomaterials 2017; 132: 1-15.
Topical lipoproteoplex-gene therapy accelerates diabetic wound closure
Untreated
Lipoproteoplex
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Elastin-like-Polypeptide (ELP):(VPGXG)n Repeating unitsSelf assembles into helical beta spiral
Cartilage Oligomeric Matrix Protein (COMPcc):Homopentameric structureHydrophobic pore
COMPcc Block polymers
Ozbek, S.,et al. EMBO J. 2002; 21: 5960.Despania J.. et al., J.Contolled release, 2016, 240, 98-108Gunasekar S.K. et al., Biochemistry, 2009, 48, 8559-8567
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Elastin (E)COMP (C)
COMPcc (C) and Elastin (E)
Ozbek, S.,et al. EMBO J. 2002; 21: 5960.Despania J.. et al., J.Contolled release, 2016, 240, 98-108Gunasekar S.K. et al., Biochemistry, 2009, 48, 8559-8567
MRGSHHHHHHGSGDL APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT VMESDAS GKLNC:[(VPGVG)2 VPGFG (VPGVG)2]5E:
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Au
Gold Nanoparticles(AuNP)
+
Protein-AuNPsassemblies
Gold Nanoparticle templationCOMPcc (C) and Elastin (E) fusion protein
Dai, M., Frezzo, J.A. et al. J. Nanomed Nanotechnol. 2016; 7:356.
Reducing agent
MRGSHHHHHHGSACELA(AT)6AACG-C-LQA(AT)6AVDKPIASSA-E-LEGSGTGGAKLNCE:
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Au
Gold Nanoparticles(AuNPs)
+
Protein-AuNPsassemblies
Gold Nanoparticle templationCOMPcc (C) and Elastin (E) fusion protein
Dai, M., Frezzo, J.A. et al. J. Nanomed Nanotechnol. 2016; 7:356.
Reducing agent
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+CCM
+CCM
Uptake of Curcumin (CCM) by MCF-7 breast cancer cells
CCM absorption at 420nm
Gold Nanoparticle and Curcumin (CCM) bindingCOMPcc (C) and Elastin (E) fusion protein
Dai, M., Frezzo, J.A. et al. J. Nanomed Nanotechnol. 2016; 7:356.
MCF-7 cells with CE MCF-7 cells with CE-AuNP
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+CCM
+CCM
Uptake of Curcumin (CCM) by MCF-7 breast cancer cells
Gold Nanoparticle and Curcumin uptakeCOMPcc (C) and Elastin (E) fusion protein
Dai, M., Frezzo, J.A. et al. J. Nanomed Nanotechnol. 2016; 7:356.
MCF-7 cells with CE MCF-7 cells with CE-AuNP
CE-CCM CE-AuNP-CCM CCM
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Elastin (E) COMP (C)COMP (C)
COMPcc (C) and Elastin (E) triblock: CEC
Olsen, A.J, et al. Biomacromolecules 2018 (accepted).
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Elastin (E) COMP (C)COMP (C)
Nanoparticles formation
COMPcc (C) and Elastin (E) triblock: CEC
Olsen, A.J, et al. Biomacromolecules 2018 (accepted).
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Elastin (E) COMP (C)COMP (C)
Nanoparticles formation Gel formation
COMPcc (C) and Elastin (E) triblock: CEC
Olsen, A.J, et al. Biomacromolecules 2018 (accepted).Gel image: Yao Wang
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SummarySmart Biomaterials
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SummaryApplications
Osteoarthritis Fluorine imaging
Gene delivery
Breast cancer
Iron templation
Biomimicry
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Protein Engineered Nanomaterials
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Protein Engineered Nanomaterials
Dr. Jin Kim Montclare Lindsay K. Hill Dr. Priya Katyal
Email: [email protected]