In this project funded by the Experimental Physical Chemistry Program in the Chemistry Division, Professor Reinhard Schweitzer-Stenner and his students will employ a battery of spectroscopic techniques to investigate the structure of unfolded peptides in water. For a long time, it was assumed that the biologically active ("native") form of most proteins involves one of the well known folded states, such as the alpha helix or beta sheet. In recent years, the discovery of native unfolded proteins has challenged the physical chemistry discipline to develop approaches to determine and understand their structure. While "unfolded," these protein systems are not completely random, but have local order or "propensities" that can, in principle, be determined with spectroscopic techniques. The first aim of the research will be to determine the intrinsic propensity of individual amino acids. Different amino acids will be inserted into a host glycine peptide chain, and examined using conventional infrared, Raman, and two dimensional nuclear magnetic resonance (2D NMR) spectroscopies, and also advanced techniques such as vibrational circular dichroism (VDC) and Raman Optical Activity (ROA) spectroscopies. In collaboration with Professor Minghaeng Cho at Korea University, the infrared, Raman and NMR spectra will be modeled using quantum mechanical and molecular dynamics simulations. The second aim is to explore the dependence of amino acid conformational propensities on the structure and charge of adjacent amino acids in the peptide chain. For example, what happens to the conformational tendencies of a given amino acid if the next acid in the chain is the branched species alanine? The third part of the project is aimed at exploring the structure of unfolded alanine based peptides. While conclusive evidence now suggests that alanine has a high preference for polyproline II like conformations, it is yet unclear whether extended conformations are maintained in longer peptides which contain between 6 and 12 amino acid residues. Finally, energy transfer experiments will be performed on specifically labeled peptides to measure end to end distance distributions.
Taken together, the expected results will substantially enhance the understanding of the properties of the unfolded state of peptides and proteins. The combination of experiment and theory will enable us (and others) to judge the validity of coil libraries for models of unfolded peptides and proteins, as well as assist computationally oriented researchers in constructing molecular dynamics force fields suitable for unfolded states. Graduate and undergraduate students, as well as high school students will be trained at the interface of chemistry, biochemistry, and molecular biology. They will learn quantum mechanics, the theory and practice of Raman, optical absorption and CD spectroscopy, protein biochemistry, and different aspects of the relationship between structure and function of proteins. Students from underrepresented groups will be involved in the project.
. Conventional wisdom says that unfolded peptides and proteins are structureless because individual segments just jump between many possible structures on a very fast timescale (picoseconds). We developed experimental strategies based on optical and nuclear magnetic resonance techniques which enabled us to determine the distribution of peptide backbone conformations wiht respect to two so-called dihedral angles, wich determine the orientation between two adjacent structural units of a polypeptide. We found that the number of conformations assumed by individual building blocks of peptides is much less than predicted by the conventional random coil model. Figure 1 shows the obtained distribution of the amino acid alanine flanked by two glycines in the investigated tripeptide. Different building blocks (i.e. different amino acid residues) show rather different distributions, which shows that the conformational manifold of unfolded peptides and proteins must be sequence dependent. We discovered hat some amino acids promote specific structures called turns which might help a peptide or protein to fold. Work on specific homopeptides revealed that conformational propeties of building blocks (amino acid residues) are substantially affected by nearest neighbors. Finally, we discovered that the addition of alcohols to the aqueous solutions of our peptides change conformational ensembles and individual thermodynamic contributions to the total Gibbs energy of the system. At some point our results will enable us to predict and analyze the structure of so called intrinsically disordered peptides and proteins which contrary to conventional wisdom perform a variety of functions in spite of the absence of any regular structure. Broader impact Scientifically, our results added substantial and important knowledge to the fast developing research on unfolded peptides and proteins. They provided unambiguous evidence for the notion that structural manifolds of these biomolecules cannot be described by the canonical random coil model. The research involved 3 graduate students, two of them graduated and left for postdoctoral appointments at prestiguous institutions. During the funding period 5 undergraduate students (all female) have worked on the project. 2 are first authors on three peer-reviewed papers (bothe supported by REU supplements), the other 3 will certainly co-author papers in the near future. Undergraduate research education of chemistry and biology majors is an integral part of the life of our research group. The PI of the group has edited a book on unfolded peptides and proteins which contains articles from all leading researchers in the field. Altogether, the project led to 13 papers in peer-reviewed journals, numerous poster presentations, and several invited talks for the PI. Thus far, our project based publications received 48 citations, the corresponding Hirsch factor is 5.
