The Dynamical Nature of Protein Folding
The folding of apomyoglobin

The Protein Folding Problem

Just how proteins fold into their final compact state from an unfolded polypeptide chain is a problem now over forty years old, dating to the work of Anfinsen, who showed that the final three dimensional structure of a protein is coded in its primary sequence. The problem is difficult theoretically because proteins are barely stable overall, and the energy of the folded structure is composed of a number of terms that are individually difficult to calculate with sufficient accuracy. In general terms, the energy landscape of the folding pathway can be viewed as a 'funnel'. The unfolded state has very high entropy while the folded state has a very low enthalpy and, as the protein folds, entropy is traded for enthalpy. The folding path can not encounter deep energy traps that would hold up the folding process. Also embedded in this view is the concept that there may not be a single pathway or a single 'transition state' in the folding dynamics. That the folding pathway is probably guided kinetically follows from the fact that the process can not involve a random search over conformation space for the lowest energy state. There are too many possible states to search in the time that proteins take to fold (the Leventhal paradox). Hence, events taking place early along the folding pathway are crucial in that they 'set-up' downstream events. The central purpose of our work in this area is to characterize experimentally the dynamics of the early stages of protein folding. We wish to determine what structures form on what time scales along the folding pathway(s) and what are the important factors for guiding the folding process. We have worked extensively on the folding of apomyoglobin.

A Picture of the Folding of Apomyoglobin

Apomyoglobin is a relatively simple, single-domain globular protein composed of 153 amino acids, with no disulfide bonds and no other secondary structure apart from the alpha helices and connecting turns. It is a model system for the folding and assembly of helices. Despite its simplicity, apoMb does not fold via two state kinetics, but rather is one of a class of proteins that fold through an intermediate having a single central compact hydrophobic core. This core consists only of a compact arrangement of the A, G, and H helices (the AGH core), whereby the two ends of the protein molecule are bound together. The core is shown in green below with the other helical structures of the protein shown as blue ribbons.

From a number of static and kinetic studies (using primarily laser induced temperature jump relaxation spectroscopy coupled with fluorescence and IR probes to follow evolving protein structure on the 50 ps to millisecond time scales; 1-3), the folding dynamics are pretty well mapped out and presented in the figure below. The formation of helical structures in isolated polypeptides that have a helical propensity form on the 50 to 200 nsec time scale, and specific runs of polypeptide of unfolded apomyoglobin also form helical like structures on the same time scale (1). The formation of the 'core' takes place along multiple pathways in 1-100 microseconds, very close to diffusion limited speed calculated for the time it takes the two ends of the protein to reach each other (5-7). The rest of the protein collapses onto the core on time scales slower than milliseconds (4).

  1. "Fast Events in Protein Folding: Helix Melting and Formation in a Small Peptide", Skip Williams, Timothy Causgrove, Rudolf Gilmanshin, Karen Fang, Robert Callender, William Woodruff, and R. Brian Dyer, Biochemistry 35, 691 (1996).
  2. "The Primary Processes of Protein Folding", Robert Callender, Rudolf Gilmanshin, Brian Dyer, and Woody Woodruff, Annual Reviews of Physical Chemistry 49, 173-202 (1998).
  3. "Infrared Studies of Fast Events in Protein Folding", R. Brian Dyer, Feng Gai, William H. Woodruff, Rudolf Gilmanshin, and Robert Callender, Accs. Chem. Res. 31, 709-716 (1998).
  4. "Fast Events In Protein Folding: Relaxation Dynamics Of Secondary And Tertiary Structure In Native Apomyoglobin", Rudolf Gilmanshin, Skip Williams, Robert H. Callender, William H. Woodruff and R. Brian Dyer, Proc. Natl. Acad. Sci. (USA) 94, 3709-3713 (1997).
  5. "The Core of Apomyoglobin E-Form: Folding at the Diffusion Limit", Rudolf Gilmanshin, Robert Callender, and R. Brian Dyer, Nature Structure Biology 5, 363-365 (1998).
  6. "Core Formation in Apomyoglobin: Probing the Upper Reaches of the Folding Energy Landscape", Miriam Gulotta, Rudolf Gilmanshin, Thomas C. Buscher, Robert Callender, and R. Brian Dyer, Biochemistry 40, 5137-5143 (2001).
  7. "Primary Folding Dynamics Of Sperm Whale Apomyoglobin: Core Formation", Miriam Gulotta, Eduard Rogatsky, Robert Callender, and R. Brian Dyer, Biophysical J. 84, 1909-1918 (2003).