A view of Protein Dynamics

The physical basis of protein structure, dynamics and function has been intensely studied for several decades. Indeed since the new millennium there has been a tremendous expansion in the number of unique topological folds that have been characterized at high resolution by crystallographic and nuclear magnetic resonance (NMR) based methods. In the midst of this rush towards a grand scale structural genomics effort, a quieter effort dedicated to the experimental characterization of protein conformational heterogeneity has also emerged. The influence of atomic scale structure on molecular recognition and catalysis by proteins is often the focus of attention while the role of dynamics is largely unknown and frequently ignored. Nevertheless, it has long been recognized that proteins are indeed dynamic systems. Weber's characterization of proteins as "kicking and screaming" is particularly poignant. However, there has been considerable frustration in obtaining comprehensive site-resolved information about protein motion.

Early insights into the time scale and character of protein internal motion largely employed local optical probes, unresolved hydrogen exchange, and one dimensional NMR techniques that, though limited, revealed a startling complexity and richness in the internal motion of proteins. These initial views contributed significantly to the development of current treatments of protein dynamics and thermodynamics. The connection to biological function rather than just biological form is more recent. Internal protein dynamics can potentially affect protein function through a variety of mechanisms, some of which are tautological or obvious in nature while others are subtle and remain to be fully explored and appreciated. There are now several examples of protein-protein and protein-ligand interactions that illustrate that dynamics may be fundamentally linked to function in several ways (see Protein Interactions). Nuclear magnetic resonance (NMR) spectroscopy is very much at the center of our current efforts to illuminate the nature of protein dynamics and their role in biological function.

Figure: The distribution of methyl order parameters (O2axis) of calmodulin side chains in complexes of calmodulin with peptides representing calmodulin-binding domains segregates into three classes: the so-called J-class, distinguished by averaging of scalar coupling constants, involves rotamer interconversion; the alpha class, distinguished by large excursions within a rotamer well (i.e. without averaging of scalar coupling constants); and the omega class that corresponds to highly restricted motion within a rotameric state.

See Igumenova et al. (2006) Chem. Rev. for more detail.

The advent of two dimensional sampling techniques in combination with appropriate isotopic enrichment strategies has allowed access to reliable NMR relaxation measurements in proteins of significant size. Our initial effort (1989) in this area was to introduce robust methods for relaxation data analysis in the context of the model-free formalism of Lipari & Szabo. Over the past decade or so we have used deuterium relaxation methods to investigate the character of methyl-bearing side chain motion in proteins. A striking clustering of methyl symmetry axis order parameters was found in the calmodulin complexes (left figure). Though sometimes obscured in other proteins, the underlying physical origin is now clear.

It would seem that Nature varies the distribution of sidechain motion across these classes. Two additional examples are shown in the figure below. Flavodoxin, a protein carrying a FMN cofactor, is unusually rigid while a 3-helix bundle is unusually dynamic. An interesting question is whether Nature has used these distributions in some way. The calmodulin system appears to suggest that this is indeed the case (see Protein Interactions).
Figure: The distribution of methyl order parameters (O2axis) for a3d, a small three-helix bundle and oxidized flavodoxin. An alpha-beta protein of moderate size with a bound FMN cofactor. Note that the a3d protein is unusually dynamic while flavodoxin is unsually rigid. It is this variation that is of prime interest as it would represent the raw material necessary for dynamic allostery.

Panel a: Fast dynamics of methyl bearing amino acids in calmodulin complexed with the calmodulin-binding domain of the smooth muscle myosin light chain kinase determined by deuterium relaxation methods. The various classes are color coded to emphasize the banding with blue corresponding to the w-class, green to the a-class and red to the J-class, as described above.

Panel b: Prediction (open circles) of the temperature dependence of the calmodulin complex using data obtained at relatively high temperature (solid circles). The open squares correspond to inelastic neutron scattering data obtained by others for ribonuclease.

See Lee & Wand (2001) Nature and Song et al. (2009) Biophys. J. for more detail.

 Some time ago we found an intriguing temperature dependence of the methyl order parameters of calmodulin in complex with the calmodulin-binding domain of the smooth muscle myosin light chain kinase. Succintly put, this data could be used to predict the so-called "glass transition" in proteins that has been observed to occur around 200 K (figure on left). This behavior has its roots in the trimodal distribution and the differential temperature dependence of the three classes of motion (panel a). The fact that this transition can be predicted using data far above the "phase transition" suggests a much simple explanation than the "solvent slaving" model that is usually invoked (panel b). See Lee & Wand (2001) Nature for more detail. This temperature dependence is recapitulated in ubiquitin in the only other similar study. See Song et al. (2009) Biophys. J.

Panels A & B: Schematic summary of the effects of pressure on the hydrogen exchange rates of amide NH of the smMLCK calmodulin-binding domain bound to calcium-saturated calmodulin. The pressure sensitivity largely arises from electrostriction effects due to the separation of ion pairs between calmodulin and the bound domain.

Panel C: Molecular interpretation of the pressure sensitivity of hydrogen exchange in the smMLCK domain bound to calmodulin. The experiment was done under conditions where the domain is always bound and thus gives a view of the molecular recognition pathway.

See Kranz et al. (2002) Biochemistry for more detail.

 We have been employing perturbation approaches to understand the degreee of coupling of motion with proteins. This is central to the idea of "dynamic allostery" proposed by Cooper several decades ago. Because NMR is so site-resolved in this context it is difficult to directly obtain information about correlated motion. Mutation, temperature and pressure are promising perturbations to illuminate coupled motion. We are fortunate in having leading-edge capability in high pressure NMR (see NMR Methods). Pressure also combines nicely with hydrogen exchange to provide a powerful insight into the statistical thermodynamic landscape of proteins. One example from Kranz et al. (2002) Biochemistry is shown on the left.

Current Project Personnel

Ellen Fu, Vignesh Kasinath, Kyle Harpole, Vonni Moorman, Nathaniel Nucci, Kathy Valentine, Sabrina Bedard, Li Liang, Fiona Love

On-Going & Future Projects

  • New NMR probes of fast motion in proteins
  • Continuing the search for correlated motion using high pressure, temperature & mutational perturbation
  • Testing of the "solvent slaving" model for protein dynamics
  • Evolutionary origins of thermodynamic "hot spots" and their entropic content
  • The dynamical nature of very high affinity complexes
  • Calculation of experimental observables using molecular dynamics and statistical thermodynamics

Useful papers

Song et al. (2007) Biophys. J.
Frederick et al. (2008) J. Phys. Chem. A
Kranz et al. (2002) Biochemistry
Igumenova et al. (2006) Chem. Rev.
Lee & Wand (2001) Nature
Lee et al. (2002) Biochemistry