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Biomedical Research in the Chen lab

SFG is well suited to studying the behavior of biological behaviors at interfaces, ranging from implanted materials to the membrane that protects a cell. Using SFG in combination with other analytical techniques- including attenuated total reflection (ATR) FTIR, four-wave mixing (FWM) spectroscopy, and AFM- we are able to obtain information on the orientation and conformation of biomolecules, to a level of detail that was previously very difficult to obtain. We can also reveal changes in the structure of a lipid bilayer brought on by interactions with molecules in solutions. Below is a summary of several sample projects underway in the Chen lab.

Orientation and conformation of biomolecules (in model cell membranes)

1. Islet amyloid polypeptide (IAPP)

IAPP plays an important role in type II diabetes. Amyloid plaques of mis-folded IAPP are found post-mortem on pancreatic islets. The first nineteen amino acids of IAPP (IAPP1-19) are responsible for interaction with cell membranes, and it is believed that this interaction is very sensitive to peptide sequence. We are applying various advanced spectroscopic techniques to investigate how IAPP interacts with cell membranes, with a goal of better understanding the mechanism of type II diabetes. We are also evaluating the effect of various drug molecules on these peptide-lipid interactions.

2. Aβ peptides

Misfolded Aβ peptides have been implicated in a variety of amyloidogenic diseases, including Alzheimer's. These peptides are the products of proteolytical cleavage of a membrane-anchored protein-amyloid precursor protein (APP), and it is thought that misfolding into β-sheet structures can be induced by interactions with the cell membrane. Until recently, limitations in available experimental techniques have complicated attempts to study the orientation of misfolded Aβ peptides. By combining multiple spectroscopic techniques, we are working to develop a systematic methodology to characterize β-sheet structures using SFG. Applied to misfolded Aβ peptides, this will improve our understanding of Alzheimer's and other amyloidogenic diseases.

3. Antimicrobial peptides (AMPs)

In the last several decades, antibiotics have become an essential tool to combat infectious diseases. However, many bacteria have developed resistance to commonly used antibiotics, posing a massive threat to public health. Various peptides (both synthetic and naturally occurring) have been observed to act directly on bacterial cell membranes, a mechanism of antibacterial action that is difficult to develop resistance against. In order to ratonally design more effective antimicrobial peptides, we must collect structural information that reveals how peptides and lipids interact. In our lab, we apply multiple spectroscopic methods to this problem, including SFG vibrational spectroscopy, double resonance SFG, four-wave mixing spectroscopy, and ATR-FTIR. Peptides that we have studied include MSI-78, magainin 2, alamethicin, melittin, pardaxin , LL-37, and the second transmembrane domain of the GABA receptor peptide.

Multiple orientations of melittin in a lipid bilayer

4. G-proteins (signal transduction)

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are a family of peripheral membrane proteins that transduce extracellular signals (e.g. light, odorants, hormones and neurotransmitters) received by G protein-coupled-receptors (GPCRs) to intracellular effector systems (e.g. ion channels and enzymes that produce second messengers). Maladaptive signaling through G proteins and their cognate receptors are involved in the progression of heart disease and hypertension, and a host of neural disorders including addiction, Parkinson’s, and schizophrenia. The pharmacological importance of heterotrimeric G proteins is underscored by the fact that over half the drugs currently on market target their signaling pathways. Heterotrimeric G proteins are comprised of Gα, Gβ, and Gγ subunits, with Gβ and Gγ forming a tightly associated dimer. We are studying Gβγ orientation, with an eye towards understanding the interactions between Gβγ and G protein coupled receptor kinase 2, Gαβγ complexes, and the activated GTP-bound Gα subunits. These measurements will enable a deeper understanding of the structures and functions of G proteins in their native environment.

Orientation of g-proteins is modulated by lipid composition

Other molecules in model membranes

1. Oligomers to treat infectious diseases

The structural complexity of many antimicrobial peptides makes them difficult to produce synthetically, and there is interest in creating simpler molecules with similar activity. By copying the most important features of antimicrobial peptides, relatively simple oligomer structures have been shown to efficiently kill bacterial cells, without harming the patient. We are studying the molecular mechanisms of interactions between such antibiotic compounds and model cell membranes. Various compounds are being studied in order to determine the features that yield potent activity without toxicity to the host.

Antimicrobial oligomer T5 acts like a molecular knife

2. Nanoparticles for drug delivery

Nanoparticles have been widely researched as drug carriers to treat many diseases including cancer. Much work has been done on the development of these nano drug-delivery vehicles, but safety and toxicity must also be considered. There exists some debate on how these nanoparticles interact with or cross cell membranes, and which parts of cells are affected. Further, even for particles known to be safe, the underlying mechanism of drug delivery, wound healing, and promotion of cell growth is not clear. We are using a variety of analytical techniques, including TEM, AFM, optical, holographic, and confocal fluorescence microscopies, sum frequency generational vibrational spectroscopy, and CARS imaging to investigate the transport of various drug-carrier nanoparticles into cells. This will provide in-depth understanding of the molecular interactions between cell membranes and nanoparticles.

Proteins at interfaces

1. Biocompatibility

Protein adsorption is the first step that occurs after a polymeric biomaterial is implanted in the body. The manner in which proteins interact with surfaces controls later processes such as cell adhesion, blood coagulation, and unnecessary immune response. We are investigating interfacial structures of important blood proteins such as FXII, fibrinogen, albumin, and kininogen using multiple techniques to understand molecular interactions between these proteins and various polymer materials, providing important clues for understanding blood compatibility.

Changes in fibrinogen structure upon adsorption

a) A few of the possible configurations of fibrinogen at the interface. The α-helix signal from each set of coiled coils is shown by solid arrows, and the net α-helix SFG signal is shown by white arrows. αC chains are not shown.
b) Schematic of fibrinogen structural changes with time after adsorption on PEU.
c) Schematic of fibrinogen structural changes with time after adsorption on SPCU or PFP.

 

2. Enzymes (Biosensing)

Biosensors play essential roles in chemical and environmental analysis, production control, fundamental studies, and clinical diagnostics. Despite the continued successes in biosensor research, some fundamental mechanistic aspects have not been satisfactorily resolved, including the activity loss of biological recognition elements, low reproducibility, and short shelf life of biosensors. A significant barrier hindering such studies is that comparatively few measurement approaches are currently available for detailed structural characterization at solid/liquid interfaces. Here we are elucidating details of protein structure (such as orientation and conformation) after immobilization on a surface by combining sum frequency generation (SFG) vibrational spectroscopic measurements with computational methods to deduce the relations between such structures and biosensor performance, providing a fundamental understanding for biosensor design and construction. Immobilized enzymes such as glutamate dehydrogenase (GDH), glucose dehydrogenase (GODH), and lactate dehydrogenase (LDH) using various immobilization methods will be studied in detail via full polarization analysis of collected spectra.

3. Surface immobilized peptides

Different immobilization methods of peptides on surfaces result in varied orientation of peptides, leading to varied functions. Here we are studying chemically immobilized peptide cercropin on polymer surfaces using different immobilization methods to optimize the orientation of cecropin, a promising antimicrobial peptide.

4. Simulations of surface adsorbed peptides

We are studying the structures of various model peptides (e.g., alpha-helical or beta-sheet peptides) physically adsorbed on polymer surfaces. These systems serve as models to develop data analysis methods for protein structural studies using vibrational spectroscopy. These results also serve as the starting point for molecular dynamics simulations. Interactions between model alpha-helices and model polymer surfaces have been studied in atomic detail using molecular dynamics simulations. Possible orientations of alpha-helical peptides can be determined and compared to experimental results, in order to better understand the factors that drive protein adsorption. Structural changes of beta-sheets interacting with polymer surfaces have also been simulated.