ata Project

Folding and Design Laboratory

Members

John Osterhout
Sunita Kulkarni

Research

alpha-t-alpha
Peptide Dissection
HX

Group Alumni

Youcef Fezoui
Tanya Knubovets
Marcela Oslin
Diane Schaak
Wujing Xian

Protein Folding/Protein Structure Discussion Group

Talk Schedule

The alpha-t-alpha Project

Protein Folding and Design Laboratory

The Concept

This project originated from a desire to elaborate on the very extensive work done to characterize monomeric helices in solution. It is clear that single helices are stabilized by short range interactions, limited usually to interactions such as salt bridges which span a turn of helix. Further, it is clear that monomeric helices are not very stable. Formation of short range structure can happen quickly and so influence the kinetic control of protein folding but in the end the low stability of monomeric helices can be overcome by longer range interactions. It is of interest then to learn about the balance of forces stabilizing helices as they interact.

At the time this project was initiated we knew that peptides corresponding to helix-turn-helix structures in proteins has been synthesized by other laboratories and that the major problem was aggregation. The "extra" hydrophobic faces in peptides derived from proteins was believed to be the culprit.

At this point we began to toy with the idea of doing a de novo design. Since a major goal of understanding protein folding is to be able to use this knowledge to design new globular proteins it seemed natural to try to design our own helix-turn-helix peptide to use as a model system in the tradition of the monomeric helical systems that had gone before.

The Design and Initial Characterization

The design of alpha-t-alpha was knowledge based which means that the design was derived as much as possible from experimental studies of monomeric peptides and coiled coils. We performed the design, synthesized the peptide and began the structural characterization primarily by NMR and CD. We published our results after we were able to observe long range NOEs between the helices and so had direct evidence that the helices were associating. Later, we published a description of the design.

De Novo Design and Structural Characterization of an alpha-Helical Hairpin Peptide - A Model System for the Study of Protein Folding Intermediates.

Fezoui, Y., Weaver, D. L. and Osterhout, J. J. (1994). Proc. Natl. Acad. Sci. USA 91, 3675-3679

Abstract

The de novo design and structural characterization of an alpha-helical hairpin peptide (a-helix/turn/a-helix, ata) is reported. The peptide is intended to provide a model system for the study of the interactions of secondary structural elements during protein folding. Both the diffusion-collision and framework models of protein folding envision that the earliest intermediates in protein folding are transient secondary structures or microdomains which interact and become mutually stabilizing. Design principles for the ata peptide were drawn from the large body of work on the structure of peptides in solution. Computer modeling was not used in the design process. Study of ata by circular dichroism and two-dimensional nuclear magnetic resonance indicates that the designed peptide is monomeric, helical and stable in aqueous solution at room temperature. Analysis of two-dimensional nuclear magnetic resonance experiments indicates that the two helices and the turn form in the intended positions and that the helices associate in the designed orientation. Development of ata represents an advance in protein design in that both the secondary structural elements and designed tertiary interactions have been realized and can be detected in solution by nuclear magnetic resonance. The resulting model system resembles a protein folding intermediate and will allow the study of interacting helices in a context that approximates an early stage in protein folding.

Commentary

At the end of this study we knew several important things. We knew from CD curves that the peptide was significantly helical. The melting curve of ata was pretty linear but the amount of CD signal certainly suggested that there was substantial helix present at room temperature. This was in contrast to monomeric helices which, in general, showed little helicity at room temperature. We had tested for aggregation by several methods. There was no concentration dependence of molar ellipticity over a range of 5 to 200 micromolar, no change in the chemical shifts of the amides between 200 micromolar and 5 millimolar, and size exclusion chromatography by HPLC gave the monomeric molecular weight. The peptide gave good NMR spectra and we were able to assign the molecule. Short and medium range NOEs were observed which showed that the positions of the alpha-helices were as designed. Finally, long range NOEs were observed between the two helices which proved helix association and were consistent with the designed interface.

Strategies and Rationales for the De Novo Design of a Helical Hairpin Peptide.

Fezoui, Y., Weaver, D. L. and Osterhout, J. J. (1995). Protein Science 4, 286-295

Abstract

The de novo design of ata, a helical hairpin peptide, is described. ata (a-helix/turn/a-helix) was developed to provide a model system for protein folding at the level of secondary structure association and stabilization. According to the prevailing models of protein folding the second step in the folding process is the association and stabilization of secondary structural elements or microdomains. A brief description of the design along with CD and NMR evidence confirming the conformation of the peptide in solution has been published (Fezoui et al., Proc. Natl. Acad. Sci, 91, 3675-3679, 1994). The present work presents a full description of the design process including the tradeoffs that were made during the development of the peptide, a discussion of recent experimental results which were not available at the time of the original design, indications of areas where, in retrospect, the design might have been done differently, and a discussion of how the present work fits into the field of de novo protein design

Structure

Solution Structure of alpha-t-alpha, a Helical Hairpin Peptide of De Novo Design.

Fezoui, Y., Connolly, P. J. and Osterhout, J. J. (1997) Protein Science 6, 1869-1877

Abstract

ata is a 38-residue peptide designed to adopt a helical hairpin conformation in solution (Fezoui et al, 1995, Protein Science 4: 286-295). A previous study of the carboxylate form of ata by CD and two-dimensional NMR indicated that the peptide was highly helical and that the helices associated in approximately the intended orientation (Fezoui et al., 1994 Proc. Natl. Acad. Sci. USA 91, 3675-3679). Here, the solution structure of ata as determined by two-dimensional NMR is reported. A total of 266 experimentally derived distance restraints and 20 dihedral angle restraints derived from J-couplings were used. One hundred initial structures were generated by distance geometry and refined by dynamical simulated annealing. Twenty three of the lowest energy structures consistent with the experimental restraints were analyzed. The results presented here show that ata is comprised of two associating helices connected by a turn region.

Cross-eyed stereo view of the superposition of the 23 final low energy structures of ata. The backbone atoms (N, Ca, and C) are rendered as blue cylinders and the carbon and sulfur atoms of the side chains of the hydrophobic interface as red cylinders. This figure is similar to Fig. 5.

Cross-eyed stereo view of the ata structure closest to the average structure. The backbone is rendered as a ribbon and the side chains of the hydrophobic interface as space filling models.

Folding and Design Laboratory Members John Osterhout Sunita Kulkarni Research alpha-t-alpha Peptide Dissection HX Group Alumni Youcef Fezoui Tanya Knubovets Marcela Oslin Diane Schaak Wujing Xian Protein Folding/Protein Structure Discussion Group Talk Schedule	The alpha-t-alpha Project Protein Folding and Design Laboratory
	The Concept This project originated from a desire to elaborate on the very extensive work done to characterize monomeric helices in solution. It is clear that single helices are stabilized by short range interactions, limited usually to interactions such as salt bridges which span a turn of helix. Further, it is clear that monomeric helices are not very stable. Formation of short range structure can happen quickly and so influence the kinetic control of protein folding but in the end the low stability of monomeric helices can be overcome by longer range interactions. It is of interest then to learn about the balance of forces stabilizing helices as they interact. At the time this project was initiated we knew that peptides corresponding to helix-turn-helix structures in proteins has been synthesized by other laboratories and that the major problem was aggregation. The "extra" hydrophobic faces in peptides derived from proteins was believed to be the culprit. At this point we began to toy with the idea of doing a de novo design. Since a major goal of understanding protein folding is to be able to use this knowledge to design new globular proteins it seemed natural to try to design our own helix-turn-helix peptide to use as a model system in the tradition of the monomeric helical systems that had gone before.
	The Design and Initial Characterization The design of alpha-t-alpha was knowledge based which means that the design was derived as much as possible from experimental studies of monomeric peptides and coiled coils. We performed the design, synthesized the peptide and began the structural characterization primarily by NMR and CD. We published our results after we were able to observe long range NOEs between the helices and so had direct evidence that the helices were associating. Later, we published a description of the design. De Novo Design and Structural Characterization of an alpha-Helical Hairpin Peptide - A Model System for the Study of Protein Folding Intermediates. Fezoui, Y., Weaver, D. L. and Osterhout, J. J. (1994). Proc. Natl. Acad. Sci. USA 91, 3675-3679 Abstract The de novo design and structural characterization of an alpha-helical hairpin peptide (a-helix/turn/a-helix, ata) is reported. The peptide is intended to provide a model system for the study of the interactions of secondary structural elements during protein folding. Both the diffusion-collision and framework models of protein folding envision that the earliest intermediates in protein folding are transient secondary structures or microdomains which interact and become mutually stabilizing. Design principles for the ata peptide were drawn from the large body of work on the structure of peptides in solution. Computer modeling was not used in the design process. Study of ata by circular dichroism and two-dimensional nuclear magnetic resonance indicates that the designed peptide is monomeric, helical and stable in aqueous solution at room temperature. Analysis of two-dimensional nuclear magnetic resonance experiments indicates that the two helices and the turn form in the intended positions and that the helices associate in the designed orientation. Development of ata represents an advance in protein design in that both the secondary structural elements and designed tertiary interactions have been realized and can be detected in solution by nuclear magnetic resonance. The resulting model system resembles a protein folding intermediate and will allow the study of interacting helices in a context that approximates an early stage in protein folding. Commentary At the end of this study we knew several important things. We knew from CD curves that the peptide was significantly helical. The melting curve of ata was pretty linear but the amount of CD signal certainly suggested that there was substantial helix present at room temperature. This was in contrast to monomeric helices which, in general, showed little helicity at room temperature. We had tested for aggregation by several methods. There was no concentration dependence of molar ellipticity over a range of 5 to 200 micromolar, no change in the chemical shifts of the amides between 200 micromolar and 5 millimolar, and size exclusion chromatography by HPLC gave the monomeric molecular weight. The peptide gave good NMR spectra and we were able to assign the molecule. Short and medium range NOEs were observed which showed that the positions of the alpha-helices were as designed. Finally, long range NOEs were observed between the two helices which proved helix association and were consistent with the designed interface. Strategies and Rationales for the De Novo Design of a Helical Hairpin Peptide. Fezoui, Y., Weaver, D. L. and Osterhout, J. J. (1995). Protein Science 4, 286-295 Abstract The de novo design of ata, a helical hairpin peptide, is described. ata (a-helix/turn/a-helix) was developed to provide a model system for protein folding at the level of secondary structure association and stabilization. According to the prevailing models of protein folding the second step in the folding process is the association and stabilization of secondary structural elements or microdomains. A brief description of the design along with CD and NMR evidence confirming the conformation of the peptide in solution has been published (Fezoui et al., Proc. Natl. Acad. Sci, 91, 3675-3679, 1994). The present work presents a full description of the design process including the tradeoffs that were made during the development of the peptide, a discussion of recent experimental results which were not available at the time of the original design, indications of areas where, in retrospect, the design might have been done differently, and a discussion of how the present work fits into the field of de novo protein design
	Structure Solution Structure of alpha-t-alpha, a Helical Hairpin Peptide of De Novo Design. Fezoui, Y., Connolly, P. J. and Osterhout, J. J. (1997) Protein Science 6, 1869-1877 Abstract ata is a 38-residue peptide designed to adopt a helical hairpin conformation in solution (Fezoui et al, 1995, Protein Science 4: 286-295). A previous study of the carboxylate form of ata by CD and two-dimensional NMR indicated that the peptide was highly helical and that the helices associated in approximately the intended orientation (Fezoui et al., 1994 Proc. Natl. Acad. Sci. USA 91, 3675-3679). Here, the solution structure of ata as determined by two-dimensional NMR is reported. A total of 266 experimentally derived distance restraints and 20 dihedral angle restraints derived from J-couplings were used. One hundred initial structures were generated by distance geometry and refined by dynamical simulated annealing. Twenty three of the lowest energy structures consistent with the experimental restraints were analyzed. The results presented here show that ata is comprised of two associating helices connected by a turn region.

	Cross-eyed stereo view of the superposition of the 23 final low energy structures of ata. The backbone atoms (N, Ca, and C) are rendered as blue cylinders and the carbon and sulfur atoms of the side chains of the hydrophobic interface as red cylinders. This figure is similar to Fig. 5.

	Cross-eyed stereo view of the ata structure closest to the average structure. The backbone is rendered as a ribbon and the side chains of the hydrophobic interface as space filling models.