Research Interests


  1. 1.  Exploring conformational ensembles of unfolded or partially folded peptides and proteins.

  2. 2.  Determining the parameters governing peptide self-aggregation.

  3. 3.  Structure and Function of heme proteins.

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

1. Exploring conformational ensembles of unfolded or partially folded peptides and proteins

It is commonly believed that the unfolded 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).  It is believed that the structure of the unfolded state reflects the propensity of the individual amino acids in the given solvent.  We have developed a mathematical algorithm by means of which the amide I band of the polarized Raman, FTIR and Vibrational Circular Dichroism spectra of peptides in solution can be utilized to determine the dihedral angles between the peptide groups in tripeptides and tetrapeptides [1,2].  This formalism was successfully applied to homo- as well as to heteropeptides [2-4].  While earlier modeling yielded representative conformations of peptide residues, we recently refined our approach by explicitly considering conformational distributions of amino acid residues in the Ramachandran space. Our structure analysis of various alanine based peptides unequivocally showed that alanine does not sample the entire sterically allowed region of the Ramachandran plot but exhibits instead a high propensity for polyproline II type conformations  [5-7]. Figure 2 shows the conformational distribution of the central residue in trialanine. However, the situation seems to change for alanine residues, which are flanked by charged residues. Such subsections seem to sample turn like conformations which yields a more compact structure of polyalanines doped with charged residues [7].

Figure 1: Polyproline II conformation adopted by tri-alanine in water


Figure 2 Conformational distribution of the central residue of trialanine.

In collaboration with the group of Harald Schwalbe at the Johann Wolfgang Goethe University ( in Frankfurt we investigated the propensities of different amino acid residues in a (nearly) context free glycine environment (e.g. GxG). To this end we combining the analysis of amide I profiles and multiple NMR J-coupling constants which exhibit different dependencies on the dihedral angles. First investigations revealed that residues with non-branched and non-aromatic side chains prefer polyproline II over -strand (leucine, glutamic acid, lycine, serine, methionine), whereas more sterically demanding residues (valine and phenylalanine) have a preference for β-strand like conformations [8-11]. Figure 3 shows a diagram displaying the fractions of PPII and β-strand for the thus far investigating peptides

Figure 3 Histogram visualizing the PPII and -strand propensities of the indicated amino acids in GxG.

Surprisingly, we found that amino acid residues with charged or polar side chains exhibit an above average sampling of structures which frequently appear in different types of β,γ and so called asx turns. A good example is protonated aspartic acid in GDG the Ramachandran plot of which is shown in Figure 4 [9,11].


Figure 4: (upper panel) Ramachandran plot protonate GDG as obtained from a combined analysis of amide I’ band profiles and J-NMR coupling constants. (lower panel) Ramachandran plot of the alanine dipeptide derived from amide I’ band profiles and J-coupling constants [12]

Recently, we collaborated with the group of Brigitta Urbanc in Drexel’s Physics Department ( to compare a amide i’/J-coupling based analysis of trialanine and the classical model system alanine dipeptide with results from MD simulations. For the latter the Urbanc group used different force fields and water models. We found that the slightly lower pPII propensity of alanine in the blocked peptide results from the absence of neighboring alanines rather them form the influence of protonable end groups.  A combination of OPLS force field and SPC/E water model produced distributions close thought not identical with our experimental results. The MD results revealed further that nearest-neighbor influence of alanine residues is mediated by changes in the hydration shell [13]. The Ramachandran plot of the alanine dipeptide is shown in Figure 5. MD simulations on GxG peptides revealed that water plays a major role in confining conformations to the left part of the upper left quadrant of the Ramachandran plot.

Siobhan Toal recently published a major study [14] that explores the influence of nearest neighbors on conformational propensities of amino acids. This project was performed as a collaboration with Prof. Harald Schwalbe’s laboratory in Frankfurt. She found that in particular residues with branched aliphatic side chains reduce peculiar conformational preferences of amino acid residues, i.e. the high polyproline II propensity of alanine. The comparatively high asx-turn propensity of aspartic acid is significant only if the amino acid residue is flanked by glycines. Moreover, she found that preferences for turn-like structures can be reduced or enhanced by nearest neighbors. Thermodynamic  data suggest that these nearest neighbor interactions are solvent mediated.


Figure 5 (left): Crystalline GAG fibrils obtained in the peptides gel phase in a 55 mol%/45 mol% ethanol/water mixture  and  6 (right). Figure 6 (right):  Crystalline GHG fibrils obtained in the peptides gel phase in water at neutral pH.

Currently, several projects are carried out in our laboratory, which are all aimed at exploring either the unfolded state of peptides. In collaboration with the Urbanc group in Physics  we explore with MD simulations to what extent polyproline II and β-strand conformations of amino acid residues are stabilized or destabilized by water. Undergraduate students Stefanie Farrell and Bridget Milorey investigate the influence of various osmolytes like glycerol, ethanol, urea, etc. on the Gibbs energy landscape of short unfolded peptides. First results revealed a rather non-linear dependence of the pPII/β equilibrium on the admixture of glycerol and ethanol. [14] Even more interesting is their discovery that GAG forms a crystalline hydrogel with fibrils in the μm range, if the peptides concentration exceeds 0.2 M and the ethanol mole fraction 55% (Figure 5) [15]. Stefanie Farrell and David DiGuiseppi currently perform experiments aimed at characterizing the gel phase and identifying the phase diagram of the ternary mixture. David DiGuiseppi currently investigates the conformational manifold of different protonation states of histidine residues in GHG.  He ended up obtaining peptide gelation as well, this time for the neutral state of GHG in water (Figure 6) [16]  Rheology measurements on GAG and GHG are being carried out in collaboration with Prof. Nicolas Alvarez in Drexel’s Department of Chemical Engineering. Two new undergraduate students (Gabrielle Lewis and Heather Carson) investigate the influence of ethanol and dimethylsulfoxide as co-solvents on spectroscopic and structural properties of short peptides


1.R. Schweitzer-Stenner. Dihedral angles of Tripeptides in Solution Determined by Polarized Raman and FTIR Spectroscopy. Biophys. J. 83, 523-532, 2002.

  1. 2.F. Eker, K. 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.

  2. 3.Eker, X. Cao, L. Nafie, K. Griebenow, and R. Schweitzer-Stenner. Peferred peptide backbone conformations in the unfolded state revealed by the structure analysis of alanine based (AXA) tripeptides in aqueous solution.  Proc. Natl. Acad. Sci. USA, 101, 10054-10059, 2004.

  3. 4.R. Schweitzer-Stenner F. Eker, K.Griebenow, X. Cao, and L. Nafie. The Conformation of Tetra-Alanine in Water determined by Polarized Raman, FT-IR and VCD Spectroscopy.  J.Am. Chem.Soc. 126, 2768-2776, 2004.

  4. 5.R. Schweitzer-Stenner, T. Measey, L. Kakalis, F. Jordan, S. Pizzanelli, C. Forte, and K. Griebenow. Conformations of Alanine Based Peptides in Water Probed by FTIR, Raman, Vibrational Circular Dichroism, Electronic Circular Dichroism, and NMR Spectroscopy. Biochemistry, 46, 1587, 2007.

  5. 6.R. Schweitzer-Stenner and T.J. Measey. The Alanine-Rich XAO Peptide Adopts a Heterogeneous Population, Including Turn Like and PPII Conformations. Proc. Natl. Acad. Sci. USA, 104, 6649-6654, 2007.

  6. 7.R. Schweitzer-Stenner. Distribution of Conformations Sampled by the Central Amino Acid Residues in Tripeptides Infered from Amide I Band Profiles and NMR Scalar Couling Constants. J. Phys. Chem. B. 113, 2922-2932, 2009.

  7. 8.A. Hagarman, T.J. Measey, D. Mathieu, H. Schwalbe and R. Schweitzer-Stenner. Intrinsic Propensities of Amino Acid Residues in GXG peptides Inferred from Amide I’ Band Profiles and NMR Scalar Coupling Constants. J. Am. Chem. Soc.132, 540-551, 2010.

  8. 9.A. Hagarman. D. Mathieu, S. Toal, T.J. Measey, H. Schwalbe, and R. Schweitzer-Stenner. Amino Acids with Hydrogen-Bonding Side Chains have an Intrinsic Tendency to sample various Turn Conformations in Aqueous Solution. Chem. Eur. J. 17, 6789-6797, 2011.

  9. 10.R. Schweitzer-Stenner, A. Hagarman, S. Toal, D. Mathieu and H. Schwalbe. Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. I. Tripeptides with long and predominantly hydrophobic side chains. Proteins 81, 955-967, 2013.

  10. 11.K. Rybka, S. Toal, D. Verbaro, D. Mathieu, H. Schwalbe and R. Schweitzer-Stenner.Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. II. Tripeptides with short side chains populating asx and β-type like turn conformations. Proteins 81, 968-983, 2013.

  11. 12. S.E. Toal, D. Meral, D.J. Verbaro, B. Urbanc and R. Schweitzer-Stenner. The pH-Independence of Trialanine and the Effects of Termini Blocking in Short Peptides: A Combined Vibrational, NMR, UVCD, and Molecular Dynamics Study. J. Phys. Chem. B. 117, 3689-3706, 2013.

  12. 13. S.E. Toal, N. Kubatova, C. Richter, V. Linhard, H. Schwalbe, and R. Schweitzer-Stenner. Randomizing the Unfolded State of peptides (and proteins) by Nearest Neighbor Interactions between Unlike Residues. Eur. J. Chem. 21, 5173-5192, 2015

  13. 14.S. Toal, A. Omidi and R. Schweitzer-Stenner. Conformational Changes of Trialanine Induced by Direct Interactions between Alanine Residues and Alcohols in Binary Mixtures of Water with Glycerol and Ethanol. J. Am. Chem. Soc. 133, 12728–12739, 2011.

  14. 15. B. Milorey, S. Farrell, S.E. Toal, and R. Schweitzer-Stenner. Demixing of water and ethanol causes conformational redistribution and gelation of the cationic GAG tripeptide. Chem. Comm. 51, 16498-16501, 2015.

  15. 16. D. DiGuiseppi and R. Schweitzer-Stenner. Probing Conformational Propensities of Histidine in Different Protonation States of the Unblocked GlycylHistidylglycine Peptide by Vibrational and NMR Spectroscopy. J. Raman Spectrosc., under review.

 2. Exploring the parameters governing peptide self-aggregation. 

We have recently discovered that amphiphatic polyalanine peptides can self-aggregate into fibrils even though the amino acid composition seems not to favor such a behavior. At certain conditions (high chloride concentration) the 16-mer (AAKA)4 can even form a hydrogel [1]. Figure 1 exhibts a Atomic Force Microscope (AFM) image of (AAKA)4 fibrils formed at conditions which do not lead to a hydrogel. At low concentrations this peptide forms aggregates instantaneously, which then decay into a (most likely monomeric) disordered state on the time scale of hours. With increasing concentration, however, the initial aggregates convert into a more stable form of aggregation, which is most likely associated with a higher degree of fibrilization. All these results depart from what is currently known about self-aggregating peptides. Self-aggregation normally requires alternating charges or alternating hydrophobic and hydrophilic residues. Our peptide do not fall into any of these categories. In collaboration with Jian-Min Yuan of Drexel Physics Department ( we carried out extensive MD simulations, which indeed revealed the formation of e.g. trimers at high peptide concentration, which decay at high temperature, in agreement with experimental results [2]. Figure 2 shows the trimer obtained from the MD simulations.

Figure 1: AFM image of (AAKA)4 (collaboration with the late Prof. Guoling Yang, Department of Physics).

Figure 2: (AAKA)4 trimer obtained from MD simulations.

In addition, we found that e.g. AAKAAAY can form fibril like aggregates. This project is carried out in collaboration with Sean Decatur from Kenyon College.T his propensity seems to depend on the presence of the C-terminal tyrosine residue. The most surpriing result was the discovery of a strongly enhanced VCD signal of the aggregated peptide’s amide I vibration [3].  We developed a theoretical model which explained this enhancement as a result of the left-handed helical twist of long fibrils which are formed by stacked parallel β-sheets of the peptide [4].

We are currently in the process of determining the parameters which causes (AAKA)4 to aggregate into soluble aggregate and into a hydrogel. We are planning to investigate how the choice of the charged residue (lysine in our case) affects the propensity for aggregation. In addition, we conduct  experiments to determine the register of AAKAAY. The undergraduate student Jodi Kraus discovered rather substantial spectroscopic changes induced in the pre-gelation of the peptide.. Our new graduate student David DiGuiseppi will get involved in the project in the near future.  Rheology measurements will be carried out in collaboration with Prof. Nicolas Alvarez in Drexel’s Department of Chemical Engineering.



1.T. Measey and R. Schweitzer-Stenner. Aggregation of the amphiphatic peptides (AAKA)n into antiparallel β-sheets. J. Am. Chem. Soc. (Communication), 128, 13324-13325, 2006.

  1. 2.S. Jang, J-M. Yuan, J. Shin, T. J. Measey, R. Schweitzer-Stenner, and F-Y. Li. Energy Landscapes Associated with the Self-Aggregation of an Alanine-Based Oligopeptide (AAKA). J. Phys. Chem. B. 113, 6054-6061, 2009.

  2. 3.T.J. Measey, K. Smith, S. Decatur, L. Zhao, G. Yang and R. Schweitzer-Stenner. The Self-aggregation of A Polyalanine Octamer Promoted by Its C-Terminal Tyrosine And Probed By A Strongly Enhanced VCD Signal. J. Am. Chem. Soc. (communication), 131, 18218-18219, 2009. 

  3. 4.T.J. Measey and R. Schweitzer-Stenner. Vibrational Circular Dichroism as a Probe of Fibrillogenesis: The Origin of the Anomalous Intensity Enhancement of the Amide I Signal of Amyloid-like Fibrils. J. Am. Chem Soc. 133, 1066-1076, 2011.

3. Structure and Function of Heme Proteins

Cytochrome c is a classical heme protein (Figure 1), which has been thoroughly investigated over more than 60 years. It is generally described as an electron transfer protein, which facilitates the electron transfer between two major membrane proteins of the mitochondria. However, evidence has now been provided that cytochrome c is a multi-functional protein, which plays a pivotal role in the initial phase of apoptosis. It has also become clear that non-native, partially unfolded states of the protein are functionally more relevant that the so-called native state which the protein adopts at neutral pH and room temperature.

Figure 1: Structure of cytochrome c (PDB: 1HRC)

Figure 2:  Structure of the heme environment of cytochrome c

We have uses circular dichroism (CD) spectroscopy to probe various non–native conformations of ferricytochrome c in solutions. Visible CD probes the interactions between the functional heme group and its protein environment.  The CD signal of the dominant B-band, which arises from a π-π* transition of the heme chromophore. It is a couplet for the native state of the protein, which is
diagnostic of an excited state splitting caused by the internal electric field in the heme pocket [1,2]. The couplet is first modified at pH 9 when the protein adopts a slightly perturbed state at which the axial ligand is still intact [3]. At pH 10, the Fe-Met 80 ligand (Figure 2) is broken and replaced by a lysine residue. This is generally called the alkaline state. In this state the CD signal of the B-band is a positive Cotton band [4], which is indicatives of a reduced band splitting. The very existence of the CD signal, however, suggests that the heme environment is still to a major extent intact.

Figure 2: CD spectra of ferricytochrome c measured at the indicated pH and low ionic strength

Recently, we found that a week long incubation of oxidized cytochrome c prevents a quick refolding of the protein once it is subjected again to neutral pH and room temperature. Instead a metastable misfolded state is formed in which the heme is most likely ligated by a hydroxyl ion. Upon protonation at rather acidic pH, a pentacoordinated quantum mixed state is formed.[4]. The protein is predominantly monomeric at micromolar concentrations but forms dimers and trimers at sub-millimolar concentration, mostly due to domain swapping. The conditions for the stabilization of this state is currently being explored by undergraduate students Valeryia Pratasava and Dmitry Malyshka. We also performed a thermodynamic analysis of the thermal unfolding of different protonation states of ferricytochrome c. This project has been started by graduate student Jonathan Soffer, who just defended his PhD thesis.

Another focus of our research on cytochrome c is the protein’s interaction with anionic cardiolipin containing liposomes by CD, absorption and resonance Raman spectroscopy. The specific aim of this project is to fully characterize structure and energy landscape of oxidized and reduced cytochrome c after the binding to the surface of such liposomes and after dissociation form the surface.  These liposome serve as a model for the innermembrane of mitochondria. This will lead us to understand why the protein gains peroxidase activity on membrane surface if challenged with hydrogen peroxide. A detailed study aimed at exploring the binding and subsequent conformational changes of ferricytochrome c to cardiolipin containing liposomes have revealed two protein binding sites. For liposomes with 20% cardiolipin binding via site 1 is insensitive to the addition of NaCl and mostly preserves the native state, including the Fe-M80 linkage. Binding via site 2, however, causes a conformational transition into a non-native, partially unfolded state [5,6,7]. This work has been carried out by former graduate student Leah Pandiscia. Our former undergraduate student Lee Serpas investigated the binding of reduced cytochrome c to CL-containing liposomes, a subject that has drawn limited attention in the cytochrome c community. He found that the protein undergoes complete oxidation upon binding to cardiolipin liposomes under aerobic conditions. This indicates a lowering of the barrier for an electron transfer between O2- and the reduced protein. The third  group member working on cytochrome is undergraduate student Dmitry Malyshka. He recently showed that the phosphate groups of cardiolipin are totally protonated even if the CL-content of lipsomes is 100% [8]. He currently employs resonance Raman spectroscopy to selectively probe partially unfolded states of cardiolipin bound cytochrome c. Bridget Milorey investigates the pH dependence of ferri- and ferrocytochrome c to CL/DOPC liposomes.  V


  1. 1.R. Schweitzer-Stenner. The Internal Electric Field in Cytochrome C Explored by Visible Electronic Circular Dichroism Spectroscopy. J. Phys. Chem. B., 112, 10358-10366, 2008. 

  2. 2.D. Verbaro, A. Hagarman, J. Soffer, and R. Schweitzer-Stenner. The pH Dependence of the 695 nm Charge Transfer Band Reveals the Population of an Intermediate State of the Alkaline Transition of Ferricytochrome c at Low Ion Concentrations. Biochemistry, 48, 2990-2996, 2009.

  3. 3.A. Hagarman, L. Duitch and R. Schweitzer-Stenner. The Conformational Manifold of Ferricytochrome c Explored by Visible and Far-UV Electronic Circular Dichroism Spectroscopy. Biochemistry, 47, 9667-9677, 2008. 

  4. 4.J.B. Soffer, E. Fradkin, L. A. Pandiscia and R. Schweitzer-Stenner. The (Not Completely Irreversible) Population of a Misfolded State of Cytochrome c under Folding Conditions. Biochemistry, 52, 1397-1408, 2013

  5. 5.L. A. Pandicia and R. Schweitzer-Stenner. Salt as a Catalyst in the Mitochondria: Returning Cytochrome c to its Native State after it Misfolds on the Surface of Cardiolipin Containing Membranes. Chem. Comm. 50, 3674-3676, 2014.

  6. 6.L.A. Pandiscia and R. Schweitzer-Stenner.Coexistence of native-like and Non-Native partially Unfolded Ferricytochrome c on the Surface of Cardilipin-Containing Liposomes. J. Phys. Chem. B. 119, 1332-1349, 2015.

  7. 7.L.A. Pandiscia and R. Schweitzer-Stenner. Coexistence of Native-Like and Non-Native Cytochrome c on Anionic Liposomes with Different Cardiolipin Content. J. Phys. Chem. B., 119, 12846-12859, 2015.

  8. 8.D. Malyshka, L.A. Pandiscia, and R. Schweitzer-Stenner. Cardiolipin Contaiing Liposomess are Fully Ionized at Physiological pH. A FT-Infrared Study on Phosphate Group Ionization. Vib. Spectrosc. 75, 86-92, 2014.

4.   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].  We explored   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.  We have recently shown that co-clustering of FceRI and MAFA (Figure 6) yields a significant reduction of the cell’s release [3,4]. This project has come to an end after Prof. Pecht’s retirement.

Figure 6. A scheme illustrating several of the possible A2-IgE-FceRI-MAFA aggregates produced by the binding of DNP-derivatized F(ab’)2 fragments of either mouse IgG or of the MAFAspecific mAb G63 to the antigen combining sites of a DNP-specific IgE and/or MAFA epitopes.  The different aggregates are characterized by the vector like representations [r; l; mfm ,mfd ,mp; nc], that represent a complex with r -FceRI- IgE receptors, l DNP-conjugated ligands, mfm MAFAmonomers and mfd MAFA dimers coclustered within a given FceRI-IgE aggregate by means of the divalent G63F(ab’)2DNPn ligands and MAFA pre-aggregated with the FceRI (mp).  nc=0, 1 denotes open and closed ring aggregates, respectively (taken from ref. 4).



  1. 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. 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. 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. 4.A. Licht, I. Pecht and R. Schweitzer-Stenner. Regulation of mast cells’ secretory response by co-clustering the Type 1 Fce receptor with the mast cell function-associated antigen. Eur. J. Immunol., 2005.