Amy R. Babbes

Visiting Assistant Professor of Chemistry

Email: [email protected]
Office: Keck Science Center 118
Phone: 909-607-8272

Educational Background
B.S., Biological Sciences (Biochemistry), Michigan Technological University
Ph.D., Medicinal Chemistry, University of Michigan
NIH Postdoctoral Fellow, The Scripps Research Institute

Courses Taught
General Chemistry, Biochemistry, Advanced Lab

Research Interests
Enzyme reaction mechanisms, characterization of novel bacterial proteins, biological oxidation-reduction reactions

Selected Publications

1. A. R. Hurshman Babbes, E. T. Powers, and J. W. Kelly. (2008). Quantification of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin and Its Disease-Associated Variants: The Relationship between Stability and Amyloidosis. Biochemistry 47: 6969-6984
   Abstract – Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to improve our understanding of the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria: dissociation of tetramers into folded monomers and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by globally fitting them to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild-type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to those of WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR, it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M TTR has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with central nervous system amyloidosis, is highly aggregation-prone and exhibits drastically reduced quaternary and tertiary structural stabilities. The observed differences in stability among the disease-associated TTR variants highlight the complexity and heterogeneity of TTR amyloid disease, an observation that has important implications for the treatment of these maladies.
   Article – https://pubs.acs.org/doi/pdf/10.1021/bi800636q
2. A. R. Hurshman, J. T. White, E. T. Powers, and J. W. Kelly. (2004). Transthyretin Aggregation Under Partially Denaturing Conditions Is a Downhill Polymerization. Biochemistry  43: 7365-7381.
   Abstract – The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or “seeds”, which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid &beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.
   Article – https://pubs.acs.org/doi/pdf/10.1021/bi049621l
3. A. R. Hurshman, C. Krebs, D. E. Edmondson, and M. A. Marletta. (2003). Ability of Tetrahydrobiopterin Analogues to Support Catalysis by Inducible Nitric Oxide Synthase: Formation of a Pterin Radical Is Required for Enzyme Activity.  Biochemistry  42: 13287-13303.
   Abstract – Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H4B) or tetrahydrobiopterin analogues (5-methyl-H4B and 4-amino-H4B), and the ability of bound 5-methyl-H4B and 4-amino-H4B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H4B forms a very stable radical, 5-methyl-H3B�, that accumulates in the arginine reaction to ~60% of the HDiNOS concentration and decays ~400-fold more slowly than H3B� (0.0003 vs 0.12 s-1). The amount of radical (5-methyl-H3B� or H3B�) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H4B-saturated FLiNOS and HDiNOS is similar to that when H4B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and �NO. A pterin radical was not observed with 4-amino-H4B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H4B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H4B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO- rather than �NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H4B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.
   Article – https://pubs.acs.org/doi/pdf/10.1021/bi035491p
4.  A. R. Hurshman and M. A. Marletta. (2002). Reactions Catalyzed by the Heme Domain of Inducible Nitric Oxide Synthase: Evidence for the Involvement of Tetrahydrobiopterin in Electron Transfer.  Biochemistry  41: 3439-3456.
   Abstract – The heme domain (iNOSheme) of inducible nitric oxide synthase (iNOS) was expressed in Escherichia coli and purified to homogeneity. Characterization of the expressed iNOSheme shows it to behave in all respects like full-length iNOS. iNOSheme is isolated without bound pterin but can be readily reconstituted with (6R)-5,6,7,8-tetrahydro-L-biopterin (H4B) or other pterins. The reactivity of pterin-bound and pterin-free iNOSheme was examined, using sodium dithionite as the reductant. H4B-bound iNOSheme catalyzes both steps of the NOS reaction, hydroxylating arginine to NG-hydroxy-L-arginine (NHA) and oxidizing NHA to citrulline and �NO. Maximal product formation (0.93 � 0.12 equiv of NHA from arginine and 0.83 � 0.08 equiv of citrulline from NHA) requires the addition of 2 to 2.5 electron equiv. Full reduction of H4B-bound iNOSheme with dithionite also requires 2 to 2.5 electron equiv. These data together demonstrate that fully reduced H4B-bound iNOSheme is able to catalyze the formation of 1 equiv of product in the absence of electrons from dithionite. Arginine hydroxylation requires the presence of a bound, redox-active tetrahydropterin; pterin-free iNOSheme or iNOSheme reconstituted with a redox-inactive analogue, 6(R,S)-methyl-5-deaza-5,6,7,8-tetrahydropterin, did not form NHA under these conditions. H4B has an integral role in NHA oxidation as well. Pterin-free iNOSheme oxidizes NHA to citrulline, N-cyanoornithine, an unidentified amino acid, and NO-. Maximal product formation (0.75 � 0.01 equiv of amino acid products) requires the addition of 2 to 2.5 electron equiv, but reduction of pterin-free iNOSheme requires only 1 to 1.5 electron equiv, indicating that both electrons for the oxidation of NHA by pterin-free iNOSheme are derived from dithionite. These data provide strong evidence that H4B is involved in electron transfer in NOS catalysis.
   Article – https://pubs.acs.org/doi/pdf/10.1021/bi012002h
5. P. Hammarstrom, X. Jiang, A. R. Hurshman, E. T. Powers, and J. W. Kelly. (2002). Sequence-Dependent Denaturation Energetics: A Major Determinant in Amyloid Disease Diversity. Proc. Natl. Acad. Sci. U.S.A.   99: 16427-16432.
   Abstract – Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease; however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V122I), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V122I TTR have nearly equivalent tetramer stabilities, V122I cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30M/T119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.
   Article – https://www.pnas.org/content/pnas/99/suppl_4/16427.full.pdf
6. A. R. Hurshman, C. Krebs, D. E. Edmondson, B. H. Huynh, and M. A. Marletta. (2000). Formation of a Pterin Radical in the Reaction of the Heme Domain of Inducible Nitric Oxide Synthase with Oxygen. Biochemistry   38: 15689-15696.
   Abstract – The heme domain (iNOS_heme) of inducible nitric oxide synthase (NOS) was expressed in Escherichia coli and purified to homogeneity. Rapid freeze−quench (RFQ) EPR was used to monitor the reaction of the reduced iNOS_heme with oxygen in the presence and absence of substrate. In these reactions, heme oxidation occurs at a rate of ∼15 s^–1 at 4 °C. A transient species with a g = 2.0 EPR signal is also observed under these conditions. The spectral properties of the g = 2.0 signal are those of an anisotropic organic radical with S = ¹/₂. Comparison of the EPR spectra obtained when iNOS_heme is reconstituted with N5-¹⁴N- and ¹⁵N-substituted tetrahydrobiopterin (H₄B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H₃B·) of the bound H₄B. Substitution of D₂O for H₂O reveals the presence of hyperfine-coupled exchangeable protons in the H₄B radical. This radical forms at a rate of 15−20 s^–1, with a slower decay rate that varies (0.12−0.7 s^–1) depending on the substrate. At 127 ms, H₃B· accumulates to a maximum of 80% of the total iNOS_heme concentration in the presence of arginine but only to ∼2.8% in the presence of NHA. Double-mixing RFQ experiments, where NHA is added after the formation of H₃B·, show that NHA does not react rapidly with H₃B· and suggest that NHA instead prevents the formation of the H₄B radical. These data constitute the first direct evidence for an NOS-bound H₃B· and are most consistent with a role for H₄B in electron transfer in the NOS reaction.
   Article – https://pubs.acs.org/doi/pdf/10.1021/bi992026c
7. M. A. Marletta, A. R. Hurshman, and K. M. Rusche. (1998). Catalysis by Nitric Oxide Synthase. Curr. Opin. Chem. Biol. 2: 656-663.
   Abstract – The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction � the hydroxylation of L-arginine.
   Article – URL not found
8. A. R. Hurshman and M. A. Marletta. (1995). Nitric Oxide Complexes of Inducible Nitric Oxide Synthase: Spectral Characterization and Effect on Catalytic Activity. Biochemistry  34: 5627-5634.
   Abstract – Nitric oxide synthase (NOS) catalyzes the oxidation of l-arginine to citrulline and nitric oxide (•NO). NOS is a hemoprotein containing a cytochrome P-450-type heme that has been shown to be involved in catalysis. It has been suggested that •NO is able to bind tightly to the heme of NOS and may in this way serve to regulate enzymatic activity. We report here the formation of both ferric and ferrous heme nitrosyl complexes with the inducible NOS from murine macrophages. The ferric nitrosyl complex is characterized by a Soret peak at 443 nm and two distinct peaks in the &alpha/&beta region at 549 and 585 nm. The ferrous nitrosyl complex has absorbance maxima at 436 and 566 nm. A transient spectral intermediate is observed under conditions of NOS turnover. This intermediate appears to be a mixture of ferric and ferrous nitrosyl complexes and is unstable in the presence of oxygen. Binding of l-arginine decreases the affinity of •NO for the ferric heme but does not appear to decrease the affinity of •NO for the ferrous heme. Addition of either oxyhemoglobin or methemoglobin to NOS assays results in a nearly 2-fold increase in enzymatic activity. This result is attributed to the ability of both forms of hemoglobin to decrease the concentration of in solution and is consistent with •NO inhibition of NOS under assay conditions. Our results show that NOS nitrosyl complexes may form under certain conditions but suggest that the relevance of such complexes to activity in vivo may be limited by their instability in an aerobic environment.
   Article – https://pubs.acs.org/doi/pdf/10.1021/bi00016a038