The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-3261 and is based on data from Edelhoch [ , Biochemistry 6:1948.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, These ~(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict E .

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI Ϸ 3.5 and a net charge of ϷϪ6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 Å from the nearest charged group. The stability of Asp49Ala is 0.5 kcal0mol greater than wild-type at pH 7 and 0.4 kcal0mol less at pH 2.5. The stability of Asp49His is 1.1 kcal0mol greater than wild-type at pH 6, where the histidine 49 side chain~pK a ϭ 7.2! is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal0mol and Glu74Lys is 1.1 kcal0mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.neurodegeneration | protein aggregation | protein misfolding | ALS | structural modeling P rotein misfolding and aggregation are linked to cell death and disease progression in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these diseases and others, the formation of amyloid plaques, often observed post mortem, has long been thought to play a role in neurodegeneration, but toxicity has never been confirmed (1-3). Recent research has shown that small, soluble oligomers, rather than insoluble amyloids, are likely to be the cytotoxic species causing neurodegeneration (4-14). These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15-18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19)(20)(21).Knowledge of the structures of these species would not only allow for definitive testing of their toxicity but could potentially lead to an understanding of disease mechanism and therapeutic strategies against diseases for which no ...

Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.spatiotemporal control | cell motility | endothelial-mesenchymal transition T he past two decades have seen a revolution in computational protein design, with remarkable milestones including design of a helical protein from first principles (1), redesign of zinc finger proteins (2), and de novo design of an α/β protein (3). These studies highlighted, as a proof of principle, our ability to rationally control the structure of proteins by using basic physical principles and phenomenology. These approaches are based on finding an optimal sequence for a given single structure or ensemble of related states, and do not provide a strategy to construct a protein capable of large on-demand conformational transitions (4, 5). A number of multistate protein design algorithms (4, 6) have been proposed; however, designing an experimentally confirmed, regulatable multistate protein, or a conformational switch (5), still remains as a challenging task because of the necessity of engineering and controlling multiple protein states (4,7,8).Such a conformational switch protein has great advantages in cell signaling, because it can be used as a universal regulatory domain (9) for precise, specific, and temporal control over rapidly activated signaling proteins (5, 10-15). Traditional genetically encoded methods for temporal protein control at the protein level have several drawbacks (5, 13). Recently developed protein switches, including derivatives of the light, oxygen, or voltage (LOV) domain (16, 17), can provide direct control at the protein level with light, but cannot be readily used in nontransparent animals. Our previous rapamycin regulated (RapR) kinase method (14) can potentially overcome this problem, but it requires expression and control of two proteins. The variable stoichiometry of these proteins renders the response more heterogeneous and essentially impractical in animals. Therefore, a single-chain, insertable, and transferable regulatory domain would be very valuable.Here we design a ligand-controlled conformational switch, uniRapR, a potentially broadly applicable, single-chai...

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1WT dimers, increasing the equilibrium dissociation constant Kd to ~10−20 μM, comparable to that of the aggressive A4V mutant. SOD1A4V is further destabilized by glutathionylation, experiencing an ~30-fold increase in Kd. Dissociation kinetics of glutathionylated SOD1WT and SOD1A4V are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1I112T has a modestly increased dissociation rate but no change in Kd when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.