Research in the Shaw laboratory is a mix of bio-inorganic chemistry, protein biophysics—with a focus on protein misfolding and amyotrophic lateral sclerosis—and a dash of medicinal chemistry and proteomics. Below you will find a brief description of some of the areas (that aren’t top secret!) that we are currently working on as of July, 2020.
Electrostatic Properties of Metalloproteins
Measuring the net electrostatic charge (Z) of a folded protein in solution (i.e., the difference in the number of cationic and anionic functional groups) has been historically challenging because few tools exist to make such a measurement and newly developed tools such as “protein charge ladders”(1, 2) remain underutilized. A search of the research literature suggests that the net charge of only ~ 0.1 % of the 83,860 proteins in the Protein Data Bank have been measured in their folded state, at any value of pH (this estimate excludes the measurement of isoelectric points, which are poor expressions of net charge). Nevertheless, the formal net charge of a protein (i.e., the value tacitly assumed from its amino acid sequence, bound co-factors, and modifications) is used to rationalize many properties of proteins including aggregation propensity (3, 4), targets of molecular recognition (5), and redox potential. (6, 7)The principal hypothesis in this set of projects is that our poor understanding of the net charge of metalloproteins—especially those that bind multiple metal ions—has inhibited a rigorous understanding of myriad chemical processes in which they are involved, and has left too many fundamental questions unanswered. For example, will the binding of a metal ion to a protein alter its net charge by a magnitude equal to the oxidation state of the metal ion (and deprotonated ligands) or will “charge regulation”—the ability of proteins to resist changes in net charge—be a predominant effect? Can the binding of multiple metal ions to a negatively charged protein lead to the inversion or abolishing of net charge? Do single amino acid substitutions really trigger “protein misfolding” diseases by lowering the net charge of a protein by a single unit, or do these mutations lower net charge by more or less than predicted? (3, 8-10) Do metalloproteins regulate net charge when cycling between oxidation states, as do transition metals within some semiconducting crystals? (11) So many important unanswered questions! In our lab, we are using a variety of analytical tools (such as “protein charge ladders” and capillary electrophoresis) to answer these fundamental questions.
Misfolded or Mischarged?
Reductions in the formal net charge of proteins by only a single unit, caused by a single missense mutation, are now hypothesized to be sufficient—without the occurrence of any perturbation to the protein’s thermostability or structure—to accelerate the fibrillization of proteins into amyloid and trigger the onset of familial protein aggregation diseases such as, amyotrophic lateral sclerosis (ALS) (12, 13) frontotemporal dementia with Parkinsonsim, and early onset Alzheimer’s disease. (10) The most exemplary case study of this point is the set of mutations in the gene encoding superoxide dismutase-1 (SOD1) that cause ~ 25 % of familial ALS.(14) Currently, approximately 45 of the ~ 160 identified ALS-linked mutations in SOD1—which are mostly missense mutations—are expected to reduce the net negative charge of the SOD1 protein (at pH 7.4; the theoretical pI of SOD1 is between 5-6, depending upon the state of metallation and N-terminal acetylation (15)). These ~ 45 mutations (the list is constantly growing) account for > 80 % of all mutations that result in non-isoelectric amino acid substitutions, deletions, or insertions (12, 13) (i.e., very few ALS mutations increase the net negative charge of SOD1, and those that do, such as V7E also destabilize the native state of apo-SOD1 (16, 17) while others such as N139D destabilize metallated but not apo-SOD1).
The importance of net charge in the aggregation of ALS mutant SOD1 is best illustrated by the occurrence of so-called “cryptic” mutations. Although many of the charge-lowering ALS-linked amino acid substitutions to SOD1 will also lower its free energy of folding regardless of metallation state (e.g., G37R, G85R, or G93R) and/or diminish its affinity to bind Cu2+ and/or or Zn2+ (e.g., H46R)—which also destabilizes SOD1 per se—there have been several “cryptic” mutations identified that reduce the formal net charge but have no significant effect on the three dimensional structure, stability, or metal binding properties of SOD1 (e.g., D90A, E100K, D101N and N139K). (8, 17) These “cryptic” amino acid substitutions either eliminate a negatively charged functional group, and/or introduce a positively charged functional group at domains across the surface of SOD1 (i.e., in the Greek-key loop IV; the edge of b6/loop VI; the “electrostatic” loop VII, and loop V). (8) This scattering of electrostatic perturbations across the surface of SOD1—each of which lessens the anionic nature of SOD1—supports the hypothesis that “cryptic” mutations promote aggregation by lowering the net negative charge of SOD1 (or equally and alternatively, that a reduction in negative electrostatic surface potential at any location on the protein surface will promote aggregation). The scattered location of “cryptic” substitutions—and all non-isoelectric ALS-linked substitutions for that matter—does not support (a priori) the hypothesis that reductions in the negative surface potential of specific domains of the SOD1 protein is required for aggregation and ALS pathogenesis. In this set of projects we are quantifying the net charge of ALS mutant SOD1 proteins (as a function of bound metal ions and pH) and developing small molecule therapies that inhibit the aggregation of these proteins by “boosting” their net charge.
1. Gitlin I, Carbeck JD, Whitesides GM. Why are proteins charged? Networks of charge-charge interactions in proteins measured by charge ladders and capillary electrophoresis. Angewandte Chemie International Edition 2006;45:3022-60.
2. Winzor DJ. Determination of the net charge (valence) of a protein: a fundamental but elusive parameter. Analytical Biochemistry 2004;325:1-20.
3. Buell AK, Hung P, Salvatella X, Welland ME, Dobson CM, Knowles TP. Electrostatic effects in filamentous protein aggregation. Biophysical Journal 2013;104:1116-26.
4. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM. Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 2003;424:805-8.
5. Shaw BF, Schneider GF, Arthanari H, et al. Complexes of native ubiquitin and dodecyl sulfate illustrate the nature of hydrophobic and electrostatic interactions in the binding of proteins and surfactants. Journal of the American Chemical Society 2011;133:17681-95.
6. Perrin BS, Jr., Ichiye T. Characterizing the effects of the protein environment on the reduction potentials of metalloproteins. Journal of Biological Inorganic Chemistry. 2013;18:103-10.
7. Rees DC. Electrostatic influence on energetics of electron transfer reactions. Proceedings of the National Academy of Sciences USA 1985;82:3082-5.
8. Shaw BF, Moustakas DT, Whitelegge JP, Faull KF. Taking charge of proteins from neurodegeneration to industrial biotechnology. Advances in Protein Chemistry and Structural Biology 2010;79:127-64.
9. Bemporad F, De Simone A, Chiti F, Dobson CM. Characterizing intermolecular interactions that initiate native-like protein aggregation. Biophys J 2012;102:2595-604.
10. Chiti F, Calamai M, Taddei N, Stefani M, Ramponi G, Dobson CM. Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. Proceedings of the National Academy of Sciences USA 2002;99 Suppl 4:16419-26.
11. Raebiger H, Lany S, Zunger A. Charge self-regulation upon changing the oxidation state of transition metals in insulators. Nature 2008;453:763-6.
12. Sandelin E, Nordlund A, Andersen PM, Marklund SS, Oliveberg M. Amyotrophic lateral sclerosis-associated copper/zinc superoxide dismutase mutations preferentially reduce the repulsive charge of the proteins. Journal of Biological Chemistry 2007;282:21230-6.
13. Bystrom R, Andersen PM, Grobner G, Oliveberg M. SOD1 mutations targeting surface hydrogen bonds promote amyotrophic lateral sclerosis without reducing apo-state stability. Journal of Biological Chemistry 2010;285:19544-52.
14. Sheng Y, Chattopadhyay M, Whitelegge J, Valentine JS. SOD1 aggregation and ALS: role of metallation states and disulfide status. Current Topics in Medicinal Chemistry 2013 (in press).
15. Shi Y, Mowery RA, Shaw BF. Effect of metal loading and subcellular pH on net charge of superoxide dismutase-1. Journal Molecular Biology 2013 (in press).
16. Rodriguez JA, Shaw BF, Durazo A, et al. Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis. Proceedings of the Nationall Academy of Sciences USA 2005;102:10516-21.
17. Shaw BF, Valentine JS. How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein? Trends in Biochemal Sciences 2007;32:78-85.