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Dystrophin and utrophin are two muscle proteins involved in Duchenne/Becker muscular dystrophy. Both proteins use tandem calponin-homology (CH) domains to bind to F-actin. We probed the role of N-terminal CH1 and C-terminal CH2 domains in the structure and function of dystrophin tandem CH domain and compared with our earlier results on utrophin to understand the unifying principles of how tandem CH domains work. Actin cosedimentation assays indicate that the isolated CH2 domain of dystrophin weakly binds to F-actin compared to the full-length tandem CH domain. In contrast, the isolated CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain. Thus, the obvious question is why the dystrophin tandem CH domain requires CH2, when its actin binding is determined primarily by CH1. To answer, we probed the structural stabilities of CH domains. The isolated CH1 domain is very unstable and is prone to serious aggregation. The isolated CH2 domain is very stable, similar to the full-length tandem CH domain. These results indicate that the main role of CH2 is to stabilize the tandem CH domain structure. These conclusions from dystrophin agree with our earlier results on utrophin, indicating that this phenomenon of differential contribution of CH domains to the structure and function of tandem CH domains may be quite general. The N-terminal CH1 domains primarily determine the actin binding function whereas the C-terminal CH2 domains primarily determine the structural stability of tandem CH domains, and the extent of stabilization depends on the strength of inter-CH domain interactions.

Tandem calponin-homology (CH) domains are the most common actin-binding domains in proteins. However, structural principles underlying their function are poorly understood. These tandem domains exist in multiple conformations with varying degrees of inter-CH-domain interactions. Dystrophin and utrophin tandem CH domains share high sequence similarity (∼82%), yet differ in their structural stability and actin-binding affinity. We examined whether the conformational differences between the two tandem CH domains can explain differences in their stability and actin binding. Dystrophin tandem CH domain is more stable by ∼4 kcal/mol than that of utrophin. Individual CH domains of dystrophin and utrophin have identical structures but differ in their relative orientation around the interdomain linker. We swapped the linkers between dystrophin and utrophin tandem CH domains. Dystrophin tandem CH domain with utrophin linker (DUL) has similar stability as that of utrophin tandem CH domain. Utrophin tandem CH domain with dystrophin linker (UDL) has similar stability as that of dystrophin tandem CH domain. Dystrophin tandem CH domain binds to F-actin ∼30 times weaker than that of utrophin. After linker swapping, DUL has twice the binding affinity as that of dystrophin tandem CH domain. Similarly, UDL has half the binding affinity as that of utrophin tandem CH domain. However, changes in binding free energies due to linker swapping are much lower by an order of magnitude compared to the corresponding changes in unfolding free energies. These results indicate that the linker region determines primarily the structural stability of tandem CH domains rather than their actin-binding affinity.

Benzyl alcohol (BA) is the most widely used antimicrobial preservative in multidose protein formulations, and has been shown to cause protein aggregation. Our previous work on a model protein cytochrome c demonstrated that this phenomenon occurs via partial unfolding. Here, we examine the validity of these results by investigating the effect of BA on a pharmaceutically relevant protein, interferon α-2a (IFNA2). IFNA2 therapeutic formulations available on the pharmaceutical market contain BA as a preservative. Isothermal aggregation kinetics and temperature scanning demonstrated that BA induced IFNA2 aggregation in a concentration-dependent manner. With increasing concentration of BA, the apparent aggregation temperature of IFNA2 linearly decreased. Denaturant melts measured using protein intrinsic fluorescence and that of the 1-anilinonaphthalene-8-sulfonic acid dye indicated that IFNA2 stability decreased with increasing BA concentration, populating a partially unfolded intermediate. Changes in nuclear magnetic resonance chemical shifts and hydrogen exchange rates identified the structural nature of this intermediate, which correlated with an aggregation "hot-spot" predicted by computational methods. These results indicate that BA induces IFNA2 aggregation by partial unfolding rather than global unfolding of the entire protein, and is consistent with our earlier conclusions from model protein studies., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.

Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to combat the growth of microbes and bacteria. These compounds have been shown to cause protein aggregation, which leads to serious immunogenic and toxic side-effects in patients. Our earlier work on a model protein cytochrome c (Cyt c) demonstrated that APs cause protein aggregation in a specific manner. The aim of this study is to validate the conclusions obtained from our model protein studies on a pharmaceutical protein. Interferon α-2a (IFNA2) is available as a therapeutic treatment for numerous immune-compromised disorders including leukemia and hepatitis C, and APs have been used in its multi-dose formulation. Similar to Cyt c, APs induced IFNA2 aggregation, demonstrated by the loss of soluble monomer and increase in solution turbidity. The extent of IFNA2 aggregation increased with the increase in AP concentration. IFNA2 aggregation also depended on the nature of AP, and followed the order m-cresol>phenol>benzyl alcohol>phenoxyethanol. This specific order exactly matched with that observed for the model protein Cyt c. These and previously published results on antibodies and other recombinant proteins suggest that the general mechanism by which APs induce protein aggregation may be independent of the protein., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using the SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also had antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Domains are in general less stable than the corresponding full-length proteins. Human utrophin tandem calponin-homology (CH) domain seems to be an exception. Reversible, equilibrium denaturant melts indicate that the isolated C-terminal domain (CH2) is thermodynamically more stable than the tandem CH domain. Thermal melts show that CH2 unfolds at a temperature higher than that at which the full-length protein unfolds. Stopped-flow kinetics indicates that CH2 unfolds slower than the full-length protein, indicating its higher kinetic stability. Thus, the utrophin tandem CH domain may be one of the few proteins in which an isolated domain is more stable than the corresponding full-length protein.

The structural determinants of the actin binding function of tandem calponin-homology (CH) domains are poorly understood, particularly the role of individual domains. We determined the actin binding affinity of isolated CH domains from human utrophin and compared them with the affinity of the full-length tandem CH domain. Traditional cosedimentation assays indicate that the C-terminal CH2 domain binds to F-actin much weaker than the full-length tandem CH domain. The N-terminal CH1 domain is less stable and undergoes severe protein aggregation; therefore, traditional actin cosedimentation assays could not be used. To address this, we have developed a folding-upon-binding method. We refolded the CH1 domain from its unfolded state in the presence of F-actin. This results in a competition between actin binding and aggregation. A differential centrifugation technique was used to distinguish actin binding from aggregation. Low-speed centrifugation pelleted CH1 aggregates, but not F-actin or its bound protein. Subsequent high-speed centrifugation resulted in the cosedimentation of bound CH1 along with F-actin. The CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain, unlike the CH2 domain. The actin binding cooperativity between the two domains was quantitatively calculated from the association constants of the full-length tandem CH domain and its CH domains, and found to be much smaller than the association constant of the CH1 domain alone. These results indicate that the actin binding affinity of the utrophin tandem CH domain is primarily determined by its CH1 domain, when compared to that of its CH2 domain or the cooperativity between the two CH domains.

One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation 'hot-spot' reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation 'hot-spot'. The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the 'hot-spot' reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the 'hot-spot' for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others., (Copyright © 2012 Wiley Periodicals, Inc.)

Deficiency of the vital muscle protein dystrophin triggers Duchenne/Becker muscular dystrophy, but the structure-function relationship of dystrophin is poorly understood. To date, molecular structures of three dystrophin domains have been determined, of which the N-terminal actin-binding domain (N-ABD or ABD1) is of particular interest. This domain is composed of two calponin-homology (CH) domains, which form an important class of ABDs in muscle proteins. A previously determined x-ray structure indicates that the dystrophin N-ABD is a domain-swapped dimer, with each monomer adopting an extended, open conformation in which the two CH domains do not interact. This structure is controversial because it contradicts functional studies and known structures of similar ABDs from other muscle proteins. Here, we investigated the solution conformation of the dystrophin N-ABD using a very simple and elegant technique of pyrene excimer fluorescence. Using the wild-type protein, which contains two cysteines, and the corresponding single-cysteine mutants, we show that the protein is a monomer in solution and is in a closed conformation in which the two CH domains seem to interact, as observed from the excimer fluorescence of pyrene-labeled wild-type protein. Excimer fluorescence was also observed in its actin-bound form, indicating that the dystrophin N-ABD binds to F-actin in a closed conformation. Comparison of the dystrophin N-ABD conformation with other ABDs indicates that the tandem CH domains in general may be monomeric in solution and predominantly occur in closed conformation, whereas their actin-bound conformations may differ., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne, and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms., (Copyright © 2012 Wiley Periodicals, Inc.)

Multidose protein formulations require an effective antimicrobial preservative (AP) to inhibit microbial growth during long-term storage of unused formulations. m-cresol (CR) is one such AP, but it has been shown to cause protein aggregation. However, the fundamental physical mechanisms underlying such AP-induced protein aggregation are not understood. In this study, we used a model protein cytochrome c to identify the protein unfolding that triggers protein aggregation. CR induced cytochrome c aggregation at preservative concentrations that are commonly used to inhibit microbial growth. Addition of CR decreased the temperature at which the protein aggregated and increased the aggregation rate. However, CR did not perturb the tertiary or secondary structure of cytochrome c. Instead, it populated an "invisible" partially unfolded intermediate where a local protein region around the methionine residue at position 80 was unfolded. Stabilizing the Met80 region drastically decreased the protein aggregation, which conclusively shows that this local protein region acts as an aggregation "hotspot." On the basis of these results, we propose that APs induce protein aggregation by partial rather than global unfolding. Because of the availability of site-specific probes to monitor different levels of protein unfolding, cytochrome c provided a unique advantage in characterizing the partial protein unfolding that triggers protein aggregation., (Copyright © 2011 Wiley-Liss, Inc.)

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as nondisease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins., (Copyright 2010 Wiley-Liss, Inc.)

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations--L54R, A168D, A171P, and Y231N--on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded alpha-helical protein in solution, as is evident from its alpha-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR (15)N-(1)H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal (T(1)) and transverse (T(2)) relaxation experiments. Compared to WT, three mutants--L54R, A168D, and A171P--show a decreased alpha-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a "molten globule" state. In contrast, Y231N has an alpha-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-beta structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.

The mechanisms whereby volatile general anesthetics reversibly alter protein function in the central nervous system remain obscure. Using three different spectroscopic approaches, evidence is presented that binding of the modern general anesthetic sevoflurane to the hydrophobic core of a model four-alpha-helix bundle protein results in structural changes. Aromatic residues in the hydrophobic core reorient into new environments upon anesthetic binding, and the protein as a whole becomes less dynamic and exhibits structural tightening. Comparable structural changes in the predicted in vivo protein targets, such as the gamma-aminobutyric acid type A receptor and the N-methyl-D-aspartate receptor, may underlie some, or all, of the behavioral effects of these widely used clinical agents.

The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four-alpha-helix bundle (Aalpha(2)-L1M/L38M)(2). The alpha-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-alpha-helix bundle (Aalpha(2)-L38M)(2) reported earlier. The affinity for binding halothane is somewhat improved with a K(d) = 120 +/- 20 microM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-alpha-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins.