This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The 2D IR spectra of peptide modes in a variety of environments have now been examined by means of dual frequency 2D IR. In this method the two modes of interest are both incorporated into the same nonlinear signal so their joint signal exists only when they are coupled in some manner. Another approach that we have introduced is the dual isotope replacement which is a strategy for exposing structural proximities by means of 2D IR. The successful preliminary results using these methods have prompted more ambitious experiments that can answer new types of questions. Dual frequency methods are needed because the bandwidth of infrared laser pulses are too narrow to simultaneously access widely separated vibrational modes. In terms of dual frequency experiments a sufficient number of examples have been reported to make it clear that the approach has great potential but that the method is in its infancy. Only a few frequencies have been incorporated into the 2D IR experiment and mainly the strong peptide backbone modes have been accessed. The first dual frequency results with the pump/probe 2D IR method showed beautifully the coupling between the N-H and the C=O groups of dipeptides and N-methylacetamide. Results were also reported for heterodyned signals arising from peptides interacting with two frequencies. The method was applied to dipeptides and most recently to model systems that dramatized the amplification of the signal expected when weak transitions are coupled to strong ones. These experiments provide the opportunity to probe details of peptide structure and dynamics that cannot easily be accessed by conventional approaches. Not only can the individual amide modes covering a wide range of frequencies be accessed but engineered probes such as those that contain CN groups in a transparency region of water could deliver a new set of structural constraints. In another example, selective deuteration of carbon hydrogen bonds can expose C-D bonds for 2D IR dual frequency analysis as discussed in the next paragraph. It is important to develop 2D IR methods for the study of membrane proteins, which are vital components of the cell physiology and include the alpha-helical class of cell-surface receptors, ion channels, transporters and redox proteins. Many have a single transmembrane (TM) helix that associates with other TM helices to form helical bundles. These assemblies occur a variety of biological situations and also have advantages for the study of folding in membranes. Despite the strong interest in them, study of their 3D structures and their dynamics remains challenging by the inherent difficulty in growing 3D crystals suitable for X-ray diffraction and by their poor solubility for solution NMR studies. The TM domain of glycophorin A (GpA) helical dimers present a prototype system for 2D IR to address the structural basis of helix association. This domain is indicated to be responsible for protein dimerization and only a few residues compose the dimerization interface. 2D IR methods will also be configured to access features that stabilize the folded conformations of membrane proteins. In the folding of helical membrane proteins the driving forces might be dispersion force interactions and/or the strong hydrogen bonds formed in the membrane. With water-soluble proteins there are energetic costs of changing a buried non-polar side chain to a smaller side chain. Understanding of the folds of membrane proteins in micelles is just beginning to emerge. Work in this area will provide a particularly fertile avenue for future investigations using 2D IR methods on isotopically edited transmembrane helices that expose both the equilibrium dynamics and the structural arrangements of coupled residues in terms of their spatial arrangements across the membrane. Interhelical H-bonds are also important in the stabilization of helix-helix interactions and 2D IR is now known to be sensitive to interactions across hydrogen bonds Current goals within this Core project are: - Completion of 50 fs dual optical parametric oscillators to access frequencies from the O-H and N-H stretches region down to the amide-III at ca. 10 for dual frequency 2D IR. A large band width in each of the pulses will ensure that cross peak spectra can be recorded over the widest possible frequency range and that the joint correlations of the two modes can be clearly identified. - Development of dual frequency technologies for recording of proximities and couplings by 2D IR between amide-I, C-D, N-H, O-H and amide modes in soluble and trans-membrane peptides in vesicles, micelles and bicelles. - Theory and processing of the 2D IR spectra of dual isotopic edited peptides and multiple isotopomers of peptide aggregates. - Introduction of high optical density protocols to dual frequency 2D IR spectroscopy permitting the study of the weak C??H mode coupling to strong amide modes in membrane bound helix dimers.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001348-30
Application #
8362564
Study Section
Special Emphasis Panel (ZRG1-BCMB-N (40))
Project Start
2011-06-01
Project End
2012-05-31
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
30
Fiscal Year
2011
Total Cost
$130,080
Indirect Cost
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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