Research Interests

1. Structure analysis of peptides

It is commonly believed that the coil state of peptides and proteins is structurally random in that the molecules sample the entire allowed region of the Ramachandran space.  This view is based on Flory's classical independent site approach.  During the last 15 years, however, numerous papers have reported experimental and theoretical evidence for the existence of regular structural motifs in the coil state.  In this context the left handed polyproline II helix has become particularly relevant (Fig. 1).  Some researchers hypothesize that it might be a precursor structure for the formation of helices in water. It is believed that the structure of the coil state reflects the propensity of the individual amino acids in the given solvent. If this view is correct one expects that even small peptide fragments like di- and tripeptides can adopt stable structures. My research group at the University of Puerto Rico has started an initiative to check this prediction. We have developed a mathematical algorithm by means of which the polarized Raman and FTIR spectra of the amide I band can be utilized to determine the dihedral angles between the two peptide groups of a tripeptide [1].  This formalism was successfully applied to homo- as well as to heteropeptides [2-4]. Our results suggest that the coil state of non-interacting amino acid residues in water exist in two different conformations, namely the aforementioned PPII and a extended ß-strand (Fig. 1). The distribution depends on the amino acid. Moreover, we found that PPII is stabilized by its aqueous environment [3,4].
 

PPII of tri-alanine  
ß-strand of tri-alanine

Figure 1: Two conformations adopted by tri-alanine in water
 

Currently, we are working on a series of AXA tripeptides in water to understand the propensity of the central amino acid residue. We have already started to work on an extension of our model to allow the structure analysis of tetra- and pentapentides (linear and cyclic). This will bring us closer to biologically relevant peptides.  Our research is aimed at developing an algorithm which allows us to calculate the amide I band profiles in IR, Raman and Vibrational Circular Dichroism spectra of much longer peptides with 20 to 30 amino acids.  In this context we will particularly focus on the structure analysis of ß-amyloid peptides.  We will perform Time Dependent Raman measurement to study conformational transitions of peptides which are capable of forming classical secondary structures.  This research will become biomedically relevant in that we will be able to study the solution structure of peptides which are used in the drug development business.

References.

[1]  R. Schweitzer-Stenner. Dihedral angles of Tripeptides in Solution Determined by Polarized Raman and FTIR Spectroscopy. Biophys. J. 83, 523-532, 2002.
[2]  F. Eker, X. Cao, L. Nafie and R. Schweitzer-Stenner. Tripeptides Adopt Stable Structures in Water. A Combined Polarized Visible Raman, FTIR and VCD Spectroscopy Study. J. Am. Chem. Soc. 124, 14330-14341, 2002.
[3] F. Eker, X. Cao, L. Nafie, and R. Schweitzer-Stenner. The Structure of Alanine Based Tripeptides in Water and Dimethylsulfoxide Probed by Vibrational Spectroscopy. J. Phys. Chem. B., 107, 358-365, 2003.
[4] F. Eker, K. Griebenow, and R. Schweitzer-Stenner. Stable Conformations of Tripeptides in Aqueous Solution Studied by UV Circular Dichroism Spectroscopy. J. Am. Chem. Soc. 125, 1879-1885, 2003.
 

2. Analysis of functionally relevant heme distortions.

This research focus on determining different types of distortions which the protein matrix and axial ligands impose on the functional heme group in various heme proteins.  These distortions are determined by means of Polarized Resonance Raman Dispersion Spectroscopy.  This method involves the measurement and subsequent analysis of the depolarization ratio dispersion and the excitation profiles of prominent bands in the Raman spectra of heme proteins. From these data vibronic coupling matrix elements are obtained which are monotonous functions of symmetry classified static normal coordinate deformations (SNCDs)[1,2], the pattern of which have been obtained from normal mode calculations.  Two types of in-plane and three out-of-plane distortions have been shown to be of particular functional relevance, namely rhombic deformations along the pyrrole nitrogens (symmetry type B1g), triclinic deformations of the pyrrole rings (symmetry type B2g) as well as macrocycle ruffling, saddling and doming (symmetry types B1u, B2u and A2u, respectively).  Some of these deformations are depicted in Figure 2.

Figure 2a: B1g and B2g type deformation of a heme macrocycle.
 

                                                                                                                

Figure 2b: B1u and B2u type deformation of the heme macrocycle

Currently, we are investigating two classical heme proteins, namely cytochrome c and horseradish peroxidase.  With respect to cytochrome c we have started to determine the contribution of the internal electric field in the heme cavity to heme deformations and thus to the functional properties such as redox potential and the reorganization energy of the electron transfer process. In this context we will determine the in-plane and out-of-plane deformations of various natural and genetically engineered  mutants. Besides its biological relevance the project has also a strong molecular physics component in that it is aimed at clarifying the origin of the experimentally observed splitting of optical bands at low temperature [3]. The investigation on horseradish peroxidase currently focus on two issues, namely the physical reason and the biological relevance of the proposed quantum mixed spin state of the central iron atom [4] and possible interactions between substrate binding and heme distortions [5].  Moreover, we will investigate the role of the proximal and distal calcium atoms in adjusting the structural and functional properties of heme group (Figure 3, from ref. 6).

Figure 3: The structure of HRPC showing the backbone trace colored by the B factor; the scale is on the right.The heme,distal His170,and proximal His42 are rendered as yellow balls and sticks and the two calcium atoms as CPK.The proximal calcium site (top)also displays its solvent accessible surface area (blue-white mesh)computed using the Connolly algorithm (20)with a 1.4 Å probe radius using the Insight II software package (Accelerys,San Diego,CA)
 

References

[1] R. Schweitzer-Stenner. Allosteric linkage-induced distortions of the prosthetic group in haem proteins as derived by the theoretical interpretation of the depolarization ratio in resonance Raman scattering. Quart. Rev. Biophys. 22, 381-479, 1989.
[2] R. Schweitzer-Stenner. Polarized Resonance Raman dispersion spectroscopy on metalporphyrins. J. Porphyrins Phthalocyanines 5, 198-224, 2001.
[3] R. Schweitzer-Stenner and D. Bigman. Electronic and Vibronic Contributions to the Band Splitting in Optical Spectra of Heme Proteins. J. Phys. Chem. B. 105, 7064-7073, 2001.
[4] Q. Huang, K. Szigeti, J. Fidy and R. Schweitzer-Stenner. Structural Disorder of Native Horseradish Peroxidase C Probed by Resonance Raman and Low-Temperature Optical Absorption Spectroscopy. J. Phys. Chem. B. 107, 2822-2830, 2003.
[5] Q. Huang, W. Al-Azzam, K, Griebenow and R. Schweitzer-Stenner. Heme Structural Perturbations of PEG-Modified Horseradish Peroxidase C in Aromatic Organic Solvents Probed by Optical Absorption and Resonance Raman Dispersion Spectroscopy. Biophys. J. 84, 3285-3298, 2003.
[6] M. Laberge, Q. Huang, R. Schweitzer-Stenner and J. Fidy. The Endogenous Calcium Ions of Horseradish Peroxidase C Are Required to Maintain the Functional Nonplanarity of the Heme. Biophys. J. 84, 2542-2552, 2003.
 
 

3. Ligand receptor binding on the surface of mast cells.

In collaboration with the group of Prof. Israel Pecht at the Weizmann Institute of Science in Rehovot/Israel we have analyzed clustering of type I Fce-receptors on the surface of mast cells by receptor specific monoclonal antibodies [1] and by multivalent antigens which bind to receptor bound immunoglobulin E.  This clustering of receptors gives rise to a biochemical cascade which eventually leads to the secretion of stored granular and de novo  synthesized mediators of inflammation. In several papers published over the last 15 years we have provided substantial evidence for a transition into a long living conformation as the initial step of receptor activation [2].  Currently, we are working on understanding the role of a so called negative coreceptor called 'mast cell function associated antigen (MAFA) which has been shown to partially inhibit the cells' secretion upon clustering by monoclonal antibodies rised against it.  However, our most recent result provide evidence that the most effective inhibition occurs if one or more MAFAs are co-clustered to a Fce-receptor aggregate [3,4].
 

References

[1] E. Ortega, R. Schweitzer-Stenner and I. Pecht. Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells. EMBO J. 7, 4101-4109, 1988.
[2] R. Schweitzer-Stenner and I. Pecht. Parameters determining the stimulatory capacity of the type I Fce-receptor. Immunol. Lett. 68, 59-69, 1999.
[3] R. Schweitzer-Stenner, M. Engelke, A. Licht and I. Pecht. Mast cell stimulation by co-clustering the type I Fce-receptors with mast cell function-associated antigens. Immunol Lett. 68, 71-78.
[4] A. Licht, R. Schweitzer-Stenner and I. Pecht. Regulation of mast cells’ secretory response by co-clustering the Fce-receptors with the mast cell function-associated antigen. submitted for publication.
 

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