Biomedical subjects

Haeshin Lee

Publications and source records attributed to Haeshin Lee.

9 recordsLinked to original sources

PubMed2006

Single-molecule mechanics of mussel adhesion.

The glue proteins secreted by marine mussels bind strongly to virtually all inorganic and organic surfaces in aqueous environments in which most adhesives function poorly. Studies of these functionally unique proteins have revealed the presence of the unusual amino acid 3,4-dihydroxy-L-phenylalanine (dopa), which is formed by posttranslational modification of tyrosine. However, the detailed binding mechanisms of dopa remain unknown, and the chemical basis for mussels' ability to adhere to both inorganic and organic surfaces has never been fully explained. Herein, we report a single-molecule study of the substrate and oxidation-dependent adhesive properties of dopa. Atomic force microscopy (AFM) measurements of a single dopa residue contacting a wet metal oxide surface reveal a surprisingly high strength yet fully reversible, noncovalent interaction. The magnitude of the bond dissociation energy as well as the inability to observe this interaction with tyrosine suggests that dopa is critical to adhesion and that the binding mechanism is not hydrogen bond formation. Oxidation of dopa, as occurs during curing of the secreted mussel glue, dramatically reduces the strength of the interaction to metal oxide but results in high strength irreversible covalent bond formation to an organic surface. A new picture of the interfacial adhesive role of dopa emerges from these studies, in which dopa exploits a remarkable combination of high strength and chemical multifunctionality to accomplish adhesion to substrates of widely varying composition from organic to metallic.

Adhesiveness↗

PubMed2006

Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation.

The Per-Arnt-Sim (PAS) domain is a ubiquitous protein module with a common three-dimensional fold involved in a wide range of regulatory and sensory functions in all domains of life. The activation of these functions is thought to involve partial unfolding of N- or C-terminal helices attached to the PAS domain. Here we use atomic force microscopy to probe receptor activation in single molecules of photoactive yellow protein (PYP), a prototype of the PAS domain family. Mechanical unfolding of Cys-linked PYP multimers in the presence and absence of illumination reveals that, in contrast to previous studies, the PAS domain itself is extended by approximately 3 nm (at the 10-pN detection limit of the measurement) and destabilized by approximately 30% in the light-activated state of PYP. Comparative measurements and steered molecular dynamics simulations of two double-Cys PYP mutants that probe different regions of the PAS domain quantify the anisotropy in stability and changes in local structure, thereby demonstrating the partial unfolding of their PAS domain upon activation. These results establish a generally applicable single-molecule approach for mapping functional conformational changes to selected regions of a protein. In addition, the results have profound implications for the molecular mechanism of PAS domain activation and indicate that stimulus-induced partial protein unfolding can be used as a signaling mechanism.

Anisotropy↗

PubMed2003

PEGylation of G-CSF using cleavable oligo-lactic acid linkage.

A new class of methoxy-poly(ethylene glycol) (mPEG) derivatives having a cleavable group of oligo-lactic acid (OLA) was synthesized by ring opening polymerization of L-lactide using a terminal hydroxyl end of mPEG as an initiator. The synthesized mPEG derivatives had 0.8 and 3.6 lactic acid units based on the MALDI-TOF data. A terminal end group of mPEG-OLA was further activated with p-nitrophenyl chloroformate to produce mPEG-OLA-p-nitrophenyl carbonate (PC). mPEG-OLA-PC derivatives were conjugated to primary amine groups of recombinant human granulocyte-colony stimulating factor. PEGylated G-CSF conjugated with mPEG-OLA-PC derivatives consisted of native, mono-, di-, and tri-PEGylated species, and they did not show any apparent conformational changes even after PEGylation. When incubating in pH 7.4 buffer at 37 degrees C for 2 days, mPEG-OLA-G-CSF conjugates liberated mPEG by cleaving the OLA spacer, resulting in the gradual regeneration of G-CSF.

Humans↗

PubMed2003

A novel method for identifying PEGylation sites of protein using biotinylated PEG derivatives.

The identification of PEGylation sites is essential in the characterization of PEGylated therapeutic proteins. This report describes a simple and novel method of finding poly(ethylene glycol) (PEG) conjugation sites in PEGylated proteins by using a hetero-functional biotin-PEG-N-hydroxyl succinimide derivative. PEGylated lysozyme species having a biotin moiety at each PEG chain end were separated and digested by trypsin. Among the digested lysozyme fragments, biotin-terminated PEGylated peptide fragments were purified by a monomeric avidin immobilized column. Their mass was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry, directly indicating that PEG was conjugated to lysine 33, 97, 116 residues. Reversed-phase high-pressure liquid chromatography results for the PEGylated peptide fragments exhibited that PEGylation occurred preferentially at lysine 33> lysine 97> lysine 116.

Binding Sites↗

PubMed2003

N-terminal site-specific mono-PEGylation of epidermal growth factor.

PURPOSE: N-terminal site-specific mono-PEGylation of recombinant human epidermal growth factor (EGF) was accomplished using polyethyleneglycol (PEG) derivatives (Mw = 2000 and 5000) through a reactive terminal aldehyde group. METHODS: The site-specific PEG conjugation was conducted ata slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an alpha-amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF. RESULTS: The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such a cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability whe incubated with tissue homogenate. CONCLUSION: N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.

Animals↗

PubMed2002

A receptor-mediated gene delivery system using streptavidin and biotin-derivatized, pegylated epidermal growth factor.

An efficient receptor-mediated non-viral gene delivery formulation based on mono-pegylated recombinant human epidermal growth factor (EGF) was developed using a streptavidin-biotin system. Biotin-derivatized and mono-pegylated EGF was prepared by conjugating a biotin-PEG-NHS derivative to EGF and purified through a chromatographic method. Luciferase plasmid DNA and polyethylenimine (PEI) were complexed to form positively charged nanoparticles on which negatively charged streptavidin was first coated and then biotin-PEG-EGF conjugate was immobilized via streptavidin-biotin interaction. The EGF-PEG-biotin-streptavidin-PEI-DNA complexes were characterized in terms of their effective diameter and surface zeta (zeta)-potential value under various formulation conditions. The formulated complexes exhibited high transfection efficiency (approximately 10(8) in luciferase activity) with no inter-particle aggregation. This was attributed to enhanced cellular uptake of the resultant complexes via receptor-mediated endocytosis. Furthermore, in the presence of serum proteins, a slight decrease in transfection efficiency was observed due to the presence of PEG chains on the surface.

Biotin↗

PubMed2002

PEG grafted polylysine with fusogenic peptide for gene delivery: high transfection efficiency with low cytotoxicity.

For efficient gene delivery into cells, a new formulation method based on using polyethylene glycol (PEG) grafted poly(L-lysine) (PLL) and a fusogenic peptide is presented in this study. First, PEG grafted PLL (PEG-g-PLL) was complexed with DNA by controlling the polymer/DNA ratio to form negatively charged nano-particulate complexes. A positively charged fusogenic peptide, KALA, was then coated by ionic interaction onto the surface of polymer/DNA complexes to make net positively charged KALA/polymer/DNA complexes. The use of PEG-g-PLL for KALA coating significantly suppressed the aggregation of complexes due to steric stabilization effect of PEG present on the surface, while the use of PLL alone induced severe aggregation of the complexes via KALA mediated inter-particulate cross-linking. For PEG-g-PLL/DNA complexes, enhanced transfection efficiency was observed with increasing amount of KALA. This suggests that maintaining the size of DNA/polymer complexes after KALA coating plays an important role in gene transfection. KALA/DNA/PEG-g-PLL complexes exhibited lower cytotoxicity compared with other polymer/DNA complexes.

Cell Line↗

PubMed2002

Pegylated recombinant human epidermal growth factor (rhEGF) for sustained release from biodegradable PLGA microspheres.

Recombinant human epidermal growth factor (rhEGF) was conjugated with polyethylene glycol (PEG) to improve its physical stability during microencapsulation in biodegradable poly(lactic-co-glycolic acid) microspheres. rhEGF was conjugated with N-hydroxysuccimide (NHS)-derivatized methoxy-PEG (mPEG) of MW 2000 and 5000 under various reaction conditions to optimize the extent of pegylation. Pegylated rhEGF showed much enhanced physical stability against homogenization. Pegylated rhEGF was encapsulated in PLGA microspheres by a double emulsion solvent evaporation method to achieve a sustained release. Pegylated rhEGF exhibited a tri-phasic release profile with a reduced initial burst, compared with unpegylated rhEGF. This study demonstrated that protein pegylation enhanced physical stability of protein and could be a good approach to achieve a sustained protein release profile from biodegradable microspheres.

Amino Acid Sequence↗

PubMed2002

Preparation and characterization of mono-PEGylated epidermal growth factor: evaluation of in vitro biologic activity.

PURPOSE: To isolate mono-PEGylated epidermal growth factor (EGF) isoforms, identify the site of PEGylation, and evaluate the biologic activity of each isoform. METHODS: EGF was PEGylated with an NHS-PEG derivative (Mw 3,400). Mono-PEGylated EGF fraction was separated by gel-filtration HPLC and three mono-PEGylated EGF isoforms were purified by RP-HPLC. Tryptic digestion mapping of both EGF and mono-PEGylated EGF isoforms was performed to identify the PEGylation sites using RP-HPLC. The digested fragments were also analyzed by matrix-assisted laser desorption and ionization time of flight (MALDI-TOF) mass spectroscopy for further verification of the three PEG conjugation sites. The biologic activity of positional isoforms was evaluated by a cell proliferation assay and a receptor tyrosine kinase activity assay to determine the effect of PEGylation site on its activity. RESULTS: Mono-PEGylated EGF was composed of three positional isomers. Tryptic digestion mapping and MALDI-TOF analysis permitted the identification of the PEGylated site of the three isoforms at N-terminus. Lysine 28, and Lysine 48. PEG-N-terminus EGF, among the three positional isomers, showed the highest activity in a cell proliferation assay and in a receptor-binding assay. CONCLUSION: This study demonstrates that biologic activities of mono-PEGylated EGF isomers are highly dependent upon the site of PEGylation in EGF.

Binding Sites↗