This research will develop a systematic way to use combined sum frequency generation (SFG), double resonance SFG (DRSFG), attenuated total refection FTIR (ATR-FTIR), and four-wave mixing (FWM) studies on alpha-helical structures to characterize membrane peptide/protein orientation in a single lipid bilayer in situ. The methodology can deduce the absolute orientation and complicated orientation distribution for a membrane peptide or protein in a single lipid bilayer that closely resembles the real membrane environment. It can also study the effect of asymmetry in lipid bilayers on peptide orientations, and can characterize structural information of peptides with very small surface coverage. The orientations of various antimicrobial peptides in lipid bilayers obtained in this study can help to elucidate modes of actions of such peptides on membranes. Antibiotic resistance is one of the most pressing problems in medicine at present, and we believe that this research will impact the design and optimization of peptides for antimicrobial purposes. The methodology will also be applied to study orientations of subunits of G-proteins in various environments, lending unique insight into how receptors and G proteins are organized in membranes during signal transduction and providing fundamental insights into various diseases such as cardiac failure.
The specific aims are: 1. SFG studies supplemented by ATR-FTIR and FWM research can provide unique orientational information of various membrane peptides in a single lipid bilayer. These studies will lead to the determination of more detailed orientation distribution of the peptides in the membrane environment. The result here will also provide a calibration base for the studies proposed in Specific Aim 2. 2. DRSFG will be used to investigate the peptides examined in Specific Aim 1 to show that DRSFG can greatly improve the sensitivity of normal SFG. Unique structural information of membrane peptides with a very low surface concentration (peptide-lipid molar ratio<1:5,000) can be characterized using DRSFG. 3. In addition to ?-helical peptides, ?-helical structures in proteins will also be investigated to demonstrate the feasibility of determining structural information of secondary structural domains of membrane proteins and the orientation of membrane proteins using SFG, supplemented by ATR-FTIR and FWM. The G?1?2 subunit of a trimeric G-protein will be used as a model in this research.

Public Health Relevance

In this research a combination of vibrational spectroscopic techniques can provide vital orientational information regarding membrane peptides and proteins, which is difficult to obtain otherwise. Such work enables in-depth understanding of membrane orientations of antimicrobial peptides and G-proteins, providing important information to develop cures for infectious diseases, heart disease, asthma, opioid addiction, and hypertension.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081655-05
Application #
8306223
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Chin, Jean
Project Start
2008-08-01
Project End
2013-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
5
Fiscal Year
2012
Total Cost
$286,942
Indirect Cost
$90,922
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Yang, Pei; Homan, Kristoff T; Li, Yaoxin et al. (2016) Effect of Lipid Composition on the Membrane Orientation of the G Protein-Coupled Receptor Kinase 2-Gβ1γ2 Complex. Biochemistry 55:2841-8
Ding, Bei; Panahi, Afra; Ho, Jia-Jung et al. (2015) Probing Site-Specific Structural Information of Peptides at Model Membrane Interface In Situ. J Am Chem Soc 137:10190-8
Soblosky, Lauren; Ramamoorthy, Ayyalusamy; Chen, Zhan (2015) Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy. Chem Phys Lipids 187:20-33
Ding, Bei; Glukhova, Alisa; Sobczyk-Kojiro, Katarzyna et al. (2014) Unveiling the membrane-binding properties of N-terminal and C-terminal regions of G protein-coupled receptor kinase 5 by combined optical spectroscopies. Langmuir 30:823-31
Yang, Pei; Glukhova, Alisa; Tesmer, John J G et al. (2013) Membrane orientation and binding determinants of G protein-coupled receptor kinase 5 as assessed by combined vibrational spectroscopic studies. PLoS One 8:e82072
Yang, Pei; Boughton, Andrew; Homan, Kristoff T et al. (2013) Membrane orientation of G*(i)*(1)ýý(2) and G*(1)ýý(2) determined via combined vibrational spectroscopic studies. J Am Chem Soc 135:5044-51
Lyon, Angeline M; Tesmer, John J G (2013) Structural insights into phospholipase C-* function. Mol Pharmacol 84:488-500
Sterne-Marr, Rachel; Baillargeon, Alison I; Michalski, Kevin R et al. (2013) Expression, purification, and analysis of G-protein-coupled receptor kinases. Methods Enzymol 521:347-66
Ding, Bei; Soblosky, Lauren; Nguyen, Khoi et al. (2013) Physiologically-relevant modes of membrane interactions by the human antimicrobial peptide, LL-37, revealed by SFG experiments. Sci Rep 3:1854
Homan, K T; Glukhova, A; Tesmer, J J G (2013) Regulation of G protein-coupled receptor kinases by phospholipids. Curr Med Chem 20:39-46

Showing the most recent 10 out of 36 publications