Molecular Biophysics
Confinement and Crowding Effects on Protein Structure/Function
Eukaryotic intracellular spaces are incredibly complex. The protein content of the cytoplasm, for example, is known to be as high as 300-400 mg/mL, representing a volume fraction of up to 40%! The crowded environment of the cellular interior not only reduces the available volume for biomolecules to diffuse, it also creates extensive non-specific interfacial interactions, and the interplay of these factors in modulating the stability and function of proteins remains poorly understood. In vivo measurements of biomolecular processes have generally shown large deviations from measurements in bulk solution. These deviations are widely attributed to the combined influences of excluded volume (largely entropic and considered to stabilize structures and interactions) and weak, non-specific so-called quinary interactions (QI, largely enthalpic, often destabilizing). Direct in vivo measurement of the comparative influence of these factors on protein structure and function is impossible because the cellular contents are far too complicated, thus simplified experimental models have often been employed to study this problem. Intracellular crowding is typically replicated with high concentrations of large soluble polymers such as ficoll-70, dextran, polyvinylpyrrolidone or polyethyleneglycol. This approach changes both excluded volume and the nature of the interface at the same time, so dissection of the influence of excluded volume versus that of QI is particularly challenging using this method. We are using the confinement created by the RM to keep excluded volume constant while changing the nature of the surfactant interface to directly measure the influence of QI on protein stability. (supported by NSF MCB-1942957)
Protein Hydration Dynamics
Studies by the Wand group1-3 have shown that RMs permit comprehensive measurement of water mobility across the surface of encapsulated proteins by NMR via detection of protein-water NOEs. In collaboration with Peter Davies of Queens University, we are applying this approach to the study of antifreeze proteins (AFPs)4. AFPs bind specifically to the growing face of ice crystals, thereby preventing cellular damage under sub-freezing temperatures. Several classes of AFPs with widely divergent structural features have been discovered in organisms from bacteria to vertebrates, thus this group of proteins represents a fascinating case of convergent molecular evolution where proteins specifically recognize an orientation of water molecules. The Davies group and others have learned a great deal about how these proteins function, and our contribution is aimed at directly measuring the mobility of water molecules across the surface of AFPs using the RM approach to help distinguish between competing molecular models for AFP function.
1. C. Jorge, B. S. Marques, K. G. Valentine, A. J. Wand, Characterizing protein hydration dynamics using solution NMR spectroscopy. Methods Enzymol. (2019) 615, 77–101
2. Nucci, Nathaniel V.; Pometun, Maxim S.; Wand, A. Joshua. Site-resolved measurement of water-protein interactions by solution NMR. Nature Structural & Molecular Biology (2011), 18(2), 245-249
3. Nucci, Nathaniel V.; Pometun, Maxim S.; Wand, A. Joshua. Mapping the Hydration Dynamics of Ubiquitin. Journal of the American Chemical Society (2011), 133(32), 12326-12329
4. Gallo, Pamela N.; Iovine, Joseph C., Nucci, Nathaniel V. Toward comprehensive measurement of protein hydration dynamics: Facilitation of NMR-based methods by reverse micelle encapsulation. Methods. (2018) 148:146-153
Structural Biology of Biomedically Important Proteins
NMR is a powerful tool for studying protein structure and dynamics, but it is fundamentally limited by molecular tumbling. Large proteins tumble slowly enough that dipolar relaxation is insufficiently averaged leading to broadened spectral signals that prohibit interpretation of their spectra. Poor resolution and complex signals also arise from intrinsic disorder due to averaging of molecular environments. We are applying the RM approach to overcome these limitations by improving molecular tumbling of large proteins1 and by employing the confinement effect to promote folding of intrinsically disordered regions of encapsulated proteins2. We are investigating two protein systems: the hypoxia-inducible factor prolyl hydroxylases and the critical tumor suppressor protein p53.
Hypoxia-Inducible Factor Prolyl Hydroxylases
The hypoxia-inducible factor prolyl hydroxylases (PHDs) are the primary oxygen sensing molecules in the cell that regulate the cellular response to low intracellular oxygen (hypoxia).These proteins are prime targets for pharmaceutical treatment of ischemic disorders resulting from chronic circulatory problems and acute traumas such as heart attack and stroke. There are three known PHDs in humans: PHD1, PHD2, and PHD3. These proteins are known to differ considerably in amino acid sequence, size, localization, and activity, but very little is known about their 3-dimensional structures3. PHD2 has been identified as the most potent inhibitor of the hypoxic response and has therefore been the primary PHD of interest. Structures of a truncated version of this isoform have been described by X-ray crystallography in the presence of natural ligands and inhibitors4, but the structure of PHD1, PHD3, and full-length PHD2 remain unknown. We are working to determine these structures by NMR, using RMs to help these relatively large proteins (25-46 kDa) tumble faster while also reducing aggregation and reducing intrinsic disorder.
Tumor Suppressor Protein p53
p53 is perhaps the most well-known oncogenic protein and plays a critical role in tumor suppression. Beyond its obvious physiological importance, p53 is a fascinating model system for investigating intrinsic disorder and structure/function relationships. Full-length p53 is composed of six regions: (from N-terminus to C-terminus) an N-terminal transactivation domain (TAD), a proline-rich region (PRR), the globular DNA-binding core domain (p53c) composed of an immunoglobulin-like β-sandwich, a 33-amino acid flexible linker region, a tetramerization domain (TET), and a flexible C-terminal region that houses many cancer-related mutation sites. TET and p53c are well structured and have been characterized by crystallography, but the TAD and C-terminus are unstructured in solution. The TAD has been shown to undergo binding-induced folding that has also been linked to dysfunction in cancer-related mutations5. A recent NMR study of the core domain revealed modulation of protein dynamics by oncogenic mutations that suggest a functional role for dynamics in the core domain6. We are systematically analyzing point and truncation mutations of p53 in the crowded environment of RMs with variation of the RM surface character to tease apart the relationships between excluded volume, QI, protein stability, and protein dynamics using NMR heteronuclear correlation and spin relaxation techniques.
1. Nucci, Nathaniel V.; Marques, Bryan S.; Bédard, Sabrina; Dogan, Jakob; Gledhill, John M., Jr.; Moorman, Veronica R.; Peterson, Ronald W.; Valentine, Kathleen G.; Wand, Alison L.; Wand, A. Joshua. Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids. Journal of Biomolecular NMR (2011), 50(4), 421-430
2. Peterson, Ronald W.; Anbalagan, Karthik; Tommos, Cecilia, Wand A. Joshua. Forced Folding and Structural Analysis of Metastable Proteins. Journal of the American Chemical Society (2004), 126:31, 9498-9499
3. Myllyharju, J. Prolyl 4-hydroxylases, master regulators of the hypoxia response. Acta Physiol. 208, 148–165 (2013).
4. McDonough, M. A. et al. Cellular oxygen sensing: crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc. Natl. Acad. Sci. U. S. A. 103, 9814–9819 (2006).
5. Joerger, A. C. & Fersht, A. R. Structural Biology of the Tumor Suppressor p53. Annu. Rev. Biochem. 77, 557–582 (2008).
6. Bej, A., Rasquinha, J. A. & Mukherjee, S. Conformational Entropy as a Determinant of the Thermodynamic Stability of the p53 Core Domain. Biochemistry 57, 6265–6269 (2018).