Professor Stefan Franzen
North Carolina State University
Taught at Adam Mickiewicz University, March 26-30, 2007
The term papers should be approximately 5 pages (single spaced) and should contain at least 10 references to the primary literature. The 3 key references should be provided as PDF or paper articles. The paper should be submitted in both electronic and printed form. The paper will be due on March 31.
Paper topics
1. Fluorescence spectroscopy applications in DNA sequencing
2. Fluorescent resonance energy transfer studies of protein folding
3. Protein secondary structure determination by infrared and ultraviolet circular dichroism spectroscopy
4. Applications of surface plasmon resonance to the detection of protein and DNA binding
5. Fluorescence studies of membrane dynamics
6. Single molecule studies using fluorescence
7. Applications of Raman spectroscopy to bacteriorhodopsin and rhodopsin
Detailed Description of Topics
1. Fluorescence spectroscopy applications in DNA sequencing
This paper should describe the issue of determining multiple events in a capillary electrophoresis experiment. The elements of capillary electrophoresis (Applied Biosystems) should be discussed. The limit of detection should be mentioned in a quantitative manner. The problem of detecting multiple fluorescent molecules should be discussed in detail. Alternative methods using on-chip detection (Affymetrix) by microscopy should be discussed. These fluorescent methods should also be discussed in a quantitative manner. The problem of false positives and methods for fluorescent labeling should be mentioned.
Capillary Electrophoresis Method for DNA Sequencing
Capillary Electrophoresis and FluorescenceApplied to DNA Sequencing
2. Fluorescent resonance energy transfer and quenching studies of protein folding
This paper should describe the protein folding problem and the use of fluorescent labeling as well as intrinsic fluorescence to elucidate the distances in unfolded and partially folded states of proteins. The issues of labeling and specific problems associated with FRET pairs should be mentioned.
FRET Application to Helix Bundles
FRET Applications using Green Fluorescent Protein
FRET Applications to IOn Channels
FRET Applications in Imaging Nuclear Proteins
3. Protein secondary structure determination by infrared and ultraviolet circular dichroism spectroscopy
This paper should discuss the correlations used to obtain estimates of protein secondary structure using FTIR and UVCD methods to determine protein structure. The basic approach of principal component analysis should be described qualitatively. The concept of a spectral library should be developed. The key spectral features of both types of spectroscopy should be elucidated in figures with descriptions in the text. The methods should be compared in terms of the advantages and disadvantages of each.
Review of UVCD Protein Structural Studies
Spectroscopic Methods of Protein Secondary Structure Analysis
Holistic Approach to Protein Secondary Structure Analysis
Vibrational CD Protein Secondary Structure Analysis
Amino Acid Side Chain Spectral Analysis
4. Applications of surface plasmon resonance to the detection of protein and DNA binding
Surface plasmon resonance (SPR) is widely used to determine binding interactions of biological molecules. The Biacore instrumentation permits detection of a wide range of binding interactions of biological molecules using a polymer approach to presentation of binding sites. However, detection of monolayers has also been developed using a variety of amplification schemes (Corn, He and many others). The general methods should be discussed and specific examples of successful applications should be given.
Interference and Optical Sensing
Integrated View of Plasmon Resonance
5. Fluorescence studies of membrane dynamics
Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are two powerful methods that have been developed to observe the motion of molecules in membranes. In addition, exciplex and excimer formation can be used to observe interactions of molecules with membranes. These methods should be discussed with specific examples from cells or model systems.
6. Single molecule studies using fluorescence
Single molecule methods are of growing importance for studies of complex biological systems such as polymerase binding to DNA or the dynamics of the ribosome. The general issues associated with sensitive detection of molecules should be discussed. Total internal reflection methods should be mentioned. A specific example should be developed in detail with an example of how data analysis is performed. The information obtained from the experiment should be presented.
Single Molecule Protein Folding
Single Molecule Fluorescence Studies
7. Applications of Raman spectroscopy to bacteriorhodopsin and rhodopsin
Bacteriorhodopsin and rhodopsin are seven helix transmembrane proteins responsible for proton pumping and vision, respectively. Rhodopsin is an example of a G-protein coupled receptor. They both contain chromophores that photoisomerize. The dynamics of these molecules have been studied using Raman spectroscopy. The use of isotopic labeling to make assignments should be discussed. The use of Raman and time-resolved Raman should be mentioned with specific examples of how the changes in the Raman spectrum provide information on the time scale for isomerization.
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