Conformational Properties of Protein Denatured States

Summary

Principal Investigator: BRUCE BOWLER
Abstract: Protein denatured states remain poorly understood and yet, as the starting point for protein folding, an understanding of the conformational constraints acting upon the denatured state is central to solving the problem of how a protein folds efficiently. Misfolding diseases,such as Alzheimer's and Parkinson's diseases are major health problems, where the causative agents are believed to be non-native and denatured states of proteins. Thus, fundamental research on denatured proteins is essential to new insight into the genesis of these disease states. This laboratory has developed a novel strategy to probe the conformational and thermodynamic properties of unfolded proteins. The propensity for forming loops of different sizes is assessed through histidine-heme loop equilibria. Single surface histidine variants have been produced in yeast iso-1-cytochrome c, allowing loop equilibria for loops of 9 to 83 amino acids to be measured under denaturing conditions. Formation of closed loops are required in the earliest stages of structure accretion when a protein folds. This system has already yielded important insights into the deviation of protein denatured states from random coil behavior. Several key properties of unfolded proteins will be probed with this system. To understand how residual structure affects contact probability in a denatured protein, we will stabilize residual structure with disulfide crosslinks, insert a known stable beta hairpin into iso-1-cytochrome c, and apply our methodology to cytochrome c', which is know to have a much more compact denatured state than iso-1-cytochrome c (specific aim 1). The effect of sequence composition on denatured state conformational properties will be probed by inserting homopolymeric arnino acid sequences into the disordered N-terminal region of iso-1-cytochrome c (specific aim 2) with emphasis on the properties of the flexible amino acid glycine and the rigid amino acid proline. Kinetics studies on loop formation and breakage are planned to probe how loop size, denatured state compactness and residual structureimpact the rate at which denatured state contacts form and the factors which cause contacts to persist (specific aim 3). NMR and FRET methods will be used to correlate denatured state thermodynamic with denatured state structural properties (specific aim 4). Our multipronged approach probes key parameters of protein denatured states expectedto be principal modulators of early events in protein folding.
Funding Period: 2009-08-31 - 2010-12-31
more information: NIH RePORT

Top Publications

  1. pmc Conformational properties of polyglutamine sequences in guanidine hydrochloride solutions
    Md Khurshid Alam Khan
    Department of Chemistry and Biochemistry, and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana, USA
    Biophys J 103:1989-99. 2012
  2. pmc Scaling properties of glycine-rich sequences in guanidine hydrochloride solutions
    Michaela L Finnegan
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana, USA
    Biophys J 102:1969-78. 2012
  3. pmc Tryptophan stabilizes His-heme loops in the denatured state only when it is near a loop end
    Md Khurshid A Khan
    Department of Chemistry and Biochemistry, Biochemistry Program, and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana 59812, United States
    Biochemistry 51:3586-95. 2012
  4. pmc Residual structure in unfolded proteins
    Bruce E Bowler
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, MT 59812, USA
    Curr Opin Struct Biol 22:4-13. 2012
  5. pmc Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'
    Tanveer A Dar
    Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana 59812, United States
    Biochemistry 50:1029-41. 2011
  6. pmc Propensities of aromatic amino acids versus leucine and proline to induce residual structure in the denatured-state ensemble of iso-1-cytochrome c
    Michaela L Finnegan
    Department of Chemistry and Biochemistry, Biochemistry Program and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
    J Mol Biol 403:495-504. 2010
  7. pmc Denatured states of low-complexity polypeptide sequences differ dramatically from those of foldable sequences
    Franco O Tzul
    Department of Chemistry and Biochemistry, Biochemistry Program and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
    Proc Natl Acad Sci U S A 107:11364-9. 2010
  8. pmc Thermodynamics of loop formation in the denatured state of rhodopseudomonas palustris cytochrome c': scaling exponents and the reconciliation problem
    K Sudhindra Rao
    Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
    J Mol Biol 392:1315-25. 2009
  9. pmc Importance of contact persistence in denatured state loop formation: kinetic insights into sequence effects on nucleation early in folding
    Franco O Tzul
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, 59812, USA
    J Mol Biol 390:124-34. 2009
  10. pmc Competition between reversible aggregation and loop formation in denatured iso-1-cytochrome c
    Franco O Tzul
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana 59812, USA
    Biochemistry 48:481-91. 2009

Scientific Experts

  • BRUCE BOWLER
  • Franco O Tzul
  • Michaela L Finnegan
  • Md Khurshid A Khan
  • Md Khurshid Alam Khan
  • Tanveer A Dar
  • K Sudhindra Rao
  • Eydiejo Kurchan
  • Abbigail L Miller
  • R Dustin Schaeffer
  • Valerie Daggett
  • Arwen K Christian
  • Heinrich Roder
  • Tia N Gordon

Detail Information

Publications12

  1. pmc Conformational properties of polyglutamine sequences in guanidine hydrochloride solutions
    Md Khurshid Alam Khan
    Department of Chemistry and Biochemistry, and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana, USA
    Biophys J 103:1989-99. 2012
    ..Evaluation of Flory's characteristic ratio, C(n), for the Gln-rich and Ala-rich sequences relative to the Gly-rich sequences shows that Gln-rich sequences are stiffer than Ala-rich sequences at both 3.0 and 6.0 M GdnHCl...
  2. pmc Scaling properties of glycine-rich sequences in guanidine hydrochloride solutions
    Michaela L Finnegan
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana, USA
    Biophys J 102:1969-78. 2012
    ..The results provide a direct experimental assessment in terms of polymer properties of the distinct roles of Gly versus Ala in the folding code...
  3. pmc Tryptophan stabilizes His-heme loops in the denatured state only when it is near a loop end
    Md Khurshid A Khan
    Department of Chemistry and Biochemistry, Biochemistry Program, and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana 59812, United States
    Biochemistry 51:3586-95. 2012
    ....
  4. pmc Residual structure in unfolded proteins
    Bruce E Bowler
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, MT 59812, USA
    Curr Opin Struct Biol 22:4-13. 2012
    ....
  5. pmc Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'
    Tanveer A Dar
    Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, Montana 59812, United States
    Biochemistry 50:1029-41. 2011
    ..Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure...
  6. pmc Propensities of aromatic amino acids versus leucine and proline to induce residual structure in the denatured-state ensemble of iso-1-cytochrome c
    Michaela L Finnegan
    Department of Chemistry and Biochemistry, Biochemistry Program and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
    J Mol Biol 403:495-504. 2010
    ....
  7. pmc Denatured states of low-complexity polypeptide sequences differ dramatically from those of foldable sequences
    Franco O Tzul
    Department of Chemistry and Biochemistry, Biochemistry Program and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
    Proc Natl Acad Sci U S A 107:11364-9. 2010
    ....
  8. pmc Thermodynamics of loop formation in the denatured state of rhodopseudomonas palustris cytochrome c': scaling exponents and the reconciliation problem
    K Sudhindra Rao
    Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
    J Mol Biol 392:1315-25. 2009
    ..With regard to the reconciliation problem, it is evident that a scaling exponent consistent with a random coil is necessary but not sufficient to demonstrate random-coil behavior...
  9. pmc Importance of contact persistence in denatured state loop formation: kinetic insights into sequence effects on nucleation early in folding
    Franco O Tzul
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, 59812, USA
    J Mol Biol 390:124-34. 2009
    ..Unlike loop breakage rates, loop formation rates are insensitive to local sequence. Together, these observations suggest that contact persistence plays a more important role in defining the "folding code" than rates of loop formation...
  10. pmc Competition between reversible aggregation and loop formation in denatured iso-1-cytochrome c
    Franco O Tzul
    Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana 59812, USA
    Biochemistry 48:481-91. 2009
    ....
  11. pmc Sequence composition effects on denatured state loop formation in iso-1-cytochrome c variants: polyalanine versus polyglycine inserts
    Franco O Tzul
    Department of Chemistry and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
    J Mol Biol 371:577-84. 2007
    ..The underlying basis for the similar behavior of loops with polyalanine versus polyglycine inserts is discussed in terms of the current knowledge of the structure and loop formation kinetics of glycine versus alanine-rich peptides...
  12. ncbi Thermodynamics of protein denatured states
    Bruce E Bowler
    Department of Chemistry, University of Montana, Missoula, MT 59812, USA
    Mol Biosyst 3:88-99. 2007
    ..New insights into protein folding and novel methods to manipulate protein stability are emerging from this work...