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Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/β-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the β-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, Km and kcat, revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3–7-fold). Conversely, EGF stimulation increased kcat ∼5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of ∼5-fold in the specificity constants (kcat/Km) for EGFR peptides, whereas ∼15–40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins ∼4–6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.

Creatine kinase (CK) is a dimeric enzyme important in ATP regeneration in cells where energy demands are high. The folding of CK under equilibrium and transient conditions has been studied in detail and is found to be complex. At equilibrium in 0.8 M GuHCl, 90% of CK molecules are in the form of a partially structured, monomeric intermediate. We exploit this property to measure kinetics of refolding and unfolding to and from this equilibrium intermediate (EI), using far-UV circular dichroism and intrinsic fluorescence as structural probes. We are thus able to compare the properties of EI and the kinetic intermediate formed during the burst phase in refolding. Native CK and EI unfold with rate constants in seconds and milliseconds, respectively. As is observed for refolding of fully-denatured CK, refolding from EI to the native state shows a burst phase followed by two exponential phases. The burst phase refolding intermediate is inferred to have more structure and greater stability than the equilibrium intermediate. When refolding from the fully-denatured state in 0.8 M GuHCl, the equilibrium intermediate is formed within the dead-time of mixing in the stopped-flow apparatus. The equilibrium intermediate may thus represent a kinetic intermediate formed early during folding.

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase α2β2 complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between α subunit Asp56 and β subunit Lys167 play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an α subunit ligand, α-glycerol 3-phosphate. Arrhenius plots of the activity data under these conditions were nonlinear. The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, “open” conformation to a high activity, “closed” conformation under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The results suggest that α subunit Asp56 and β subunit Lys167 are not essential for catalysis and for allosteric communication between the α and β subunits but that their mutual interaction is important in stabilization of the active, closed form of the α2β2 complex.

Changes in the activity and conformation of Chinese hamster dihydrofolate reductase (ch-DHFR) were studied in reverse micelles of cetyltrimethylammonium bromide (CTAB) and dodecylammonium butyrate (DAB) at various water contents and denaturant concentrations. The ch-DHFR entrapped in CTAB and DAB reverse micelles shows very low activity at a lower ratio of water to surfactant (omega(0)). The activity was enhanced in the presence of low concentrations of guanidine hydrochloride. Emission fluorescence spectra of ch-DHFR in aqueous medium and in reverse micelles in the presence of low concentrations of denaturants were compared. Only a slight change in emission intensity of ch-DHFR accompanies enzyme activation in the presence of low concentrations of guanidine hydrochloride in CTAB and DAB reverse micelles. There was no detectable change in the average diameters of CTAB and DAB reverse micelles in the presence of low concentrations of denaturants measured by laser light scattering, indicating that the enzyme activation in the presence of denaturant is not due to a size change in the reverse micelles. Our results suggest that the reduced activity of ch-DHFR in reverse micelles at low omega(0) is due to the restrictions in enzyme conformation caused by an environment with low water content. The reduction in enzyme activity was restored by the presence of low concentrations of denaturant or increased water content, indicating that conformation flexibility is important for full expression of enzyme activity.

To characterize the conformational transitions that regulate the activity and specificity of the tryptophan synthase alpha 2 beta 2 complex, we have determined some effects of low concentrations of guanidine hydrochloride (GuHCl) and of urea on functional properties. We report the novel finding that GuHCl at low concentrations (0. 02-0.08 M) is a cation activator of the tryptophan synthase alpha 2 beta 2 complex. Molecular modeling studies show that GuH+ could bind at a previously identified cation binding site in the tryptophan synthase beta subunit. Addition of increasing concentrations of GuHCl has strikingly different effects on the rates of different reactions with L-serine or beta-chloro-L-alanine in the presence or absence of indole. Spectroscopic studies demonstrate that GuHCl alters the equilibrium distribution of pyridoxal 5'-phosphate intermediates formed in reactions at the active site of the beta subunit. Data analysis shows that GuHCl binds preferentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shifts the equilibrium in favor of this conformer. These results provide evidence that GuHCl exerts dual effects on tryptophan synthase as a cation, stimulating activity, and as a chaotropic agent, altering the distribution of conformational states that exhibit different reaction specificities. Our finding that the nonionic urea stabilizes the aldimine of L-serine in the presence, but not in the absence, of NaCl shows that cation binding plays an important role in the conformational transitions that regulate activity and the transmission of allosteric signals between the alpha and beta sites.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.

Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2YVMA and ERBB2G776VC, have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2YVMA to wild type using physiologically relevant peptide substrates reveals that ERBB2YVMA kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by ∼150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2YVMA phosphorylates GAB1 protein ∼70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis. Background: Activating ERBB2 mutants drive tumor formation. Results: Oncogenic ERBB2 has a striking substrate preference for GAB1 in vitro, and GAB1 hyper-phosphorylation is required for mutant ERBB2-induced cell signaling and transformation. Conclusion: Acquired substrate preference for GAB1 is critical to the ERBB2 mutant-mediated oncogenesis. Significance: Understanding the activation mechanism of mutant ERBB2s may lead to the development of therapies targeted against these oncogenic kinases.

It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028–7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulphonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.

The effect of cationic surfactants on the fluorescence of the zirconium-morin system is described. In sulphuric acid medium, the fluorescence intensity of this complex can be enhanced strongly by the cationic surfactant cetyltrimethylammonium bromide (CTAB). It was found that the decisive factor is the change in the composition of the analyte complex in the presence of CTAB. When the concentration of CTAB is near its critical micelle concentration, the absorptiometric measurement is favoured by the formation of a 1:3 [Zr(IV):morin] complex. On increasing the CTAB concentration, a mixed-ligand complex with the composition of Zr(IV):morin:SO 2− 4 = 1:1:2 is formed, and the fluorescence intensity is enhanced dramatically. The role of CTAB in the formation of the mixed-ligand complex and the enhancement of the fluorescence is discussed.

We previously reported the identification of small serine/threonine kinase (SSTK) that is expressed in postmeiotic germ cells, associates with HSP90, and is indispensable for male fertility. Sperm from SSTK-null mice cannot fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida. Here, using the yeast two-hybrid screen, we have discovered a novel SSTK-interacting protein (SIP) that is expressed exclusively in testis. The gene encoding SIP is restricted to mammals and encodes a 125-amino acid polypeptide with a predicted tetratricopeptide repeat domain. SIP is co-localized with SSTK in the cytoplasm of spermatids as they undergo restructuring and chromatin condensation, but unlike SSTK, is not retained in the mature sperm. SIP binds to SSTK with high affinity (K(d) ∼10 nM), and the proteins associate with each other when co-expressed in cells. In vitro, SIP inhibited SSTK kinase activity, whereas the presence of SIP in cells resulted in enzymatic activation of SSTK without affecting Akt or MAPK activity. SIP was found to be associated with cellular HSP70, and analyses with purified proteins revealed that SIP directly bound HSP70. Importantly, SSTK recruited SIP onto HSP90, and treatment of cells with the specific HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, completely abolished SSTK catalytic activity. Hence, these findings demonstrate that HSP90 is essential for functional maturation of the kinase and identify SIP as a cochaperone that is critical to the HSP90-mediated activation of SSTK.

Therapeutic protein products provide unique and effective treatments for numerous human diseases and medical conditions. In many cases, these treatments are used chronically to slow disease progression, reduce morbidity and/or to replace essential proteins that are not produced endogenously in patients. Therefore, any factor that reduces or eliminates the effectiveness of the treatment can lead to patient suffering and even death. One means by which efficacy of therapeutic proteins can be compromised is by an immune response, resulting in antibody-mediated neutralization of the protein’s activity or alterations in bioavailability.1,2 For example, in the case of treatment of hemophilia A, neutralizing antibodies to Factor VIII can cause life-threatening bleeding episodes, resulting in significant morbidity and necessitating treatment with a prolonged course of a tolerance-inducing therapy to reverse immunity.3,4 In other cases, drug-induced antibodies to a therapeutic version of an endogenous protein can cross-react with and neutralize the patient’s endogenous protein. If the endogenous protein serves a non-redundant biological function, such an immune response can have devastating results. For example, pure red cell aplasia can result from neutralizing antibodies to epoetin alpha. 1,2 It is well established that protein aggregates in therapeutic protein products can enhance immunogenicity2, and such an effect is therefore an important risk factor to consider when assessing product quality. The purpose of this commentary is to accomplish the following: provide brief summaries on the factors affecting protein aggregation and the key aspects of protein aggregates that are associated with immunogenicity; emphasize the current scientific gaps in understanding and analytical limitations for quantitation of species of large protein aggregates that are referred to as subvisible particles, with specific consideration of those particles 0.1–10 μm in size; offer a rationale for why these gaps may compromise the safety and/or efficacy of a product; provide scientifically sound, risked based recommendations/conclusions for assessment and control of such aggregate species. Causes of Protein Aggregation Proteins usually aggregate from partially unfolded molecules, which can be part of the native state ensemble of molecules.5 Even though product formulations are developed to maximize and maintain the fraction of the protein molecules present in the native state, significant amounts of aggregates can form, especially over pharmaceutically-relevant time scales and under stress conditions. For example, exposure to interfaces (e.g., air-liquid and solid-liquid), light, temperature fluctuations or minor impurities can induce aggregation. Such exposure can occur during processing steps, as well as in the final product container during storage, shipment and handling. Furthermore, protein particles (visible and subvisible) can be generated from protein alone or from heterogeneous nucleation on foreign micro- and nanoparticles that are shed, for example, from filling pumps or product container/closures.6–8 The levels and sizes of protein particles present in a given product can be changed by many factors relevant to commercial production of therapeutic proteins. Such factors include a change in the type of filling pump during scale-up to commercial manufacturing, changes in formulation or container/closure, and even unintentional changes in the manufacturing process such as alterations in filling pump mechanical parameters or other unforeseen factors.8,9 Thus, unless appropriate quality controls are in place for subvisible particles, a product that was safe and effective in clinical trials may unexpectedly cause adverse events in patients after commercialization. Effects of Aggregate Characteristics on Immunogenicity From work on fundamental aspects of immunology and vaccine development, it is known that large protein assemblies with repetitive arrays of antigens, in which the protein molecules have native conformation, are usually the most potent at inducing immune responses.2,10,11 Furthermore, efforts to develop more effective vaccines have shown that adsorbing antigenic proteins to nano- or microparticles comprised of other materials (e.g., colloidal aluminum salts or polystyrene) can greatly increase immunogenicity.12,13 Applying these lessons to therapeutic protein products, it has been argued that large aggregates containing protein molecules with native-like conformation pose the greatest risk of causing adverse immune responses in patients.2 Thus, for example, particles of therapeutic proteins formed by adsorption of protein molecules onto foreign micro- and nanoparticles might be particularly prone to cause immunogenicity. These particles contain numerous protein molecules, and in the two examples published to date, the adsorbed protein molecules were shown to retain their native conformations.6,8 Unfortunately, lacking are published studies that comprehensively investigate the range of parameters that could influence immunogenicity of aggregates. Because each protein may differ in aggregate formation and consequences, factors that need to be investigated include but are not limited to type, amount and size of aggregates, as well as protein conformation in aggregates, on a case by case basis. Of course, other factors, particularly pertaining to patient status and treatment protocol, are also critical in determining the propensity to generate immune responses. These include immune competence of the patients, route of administration, and dosing frequency and duration. Given the consequences of aggregate-induced immunogenicity in patients, it is important to understand these issues and to reduce the risk to product quality for every therapeutic protein product. Because the exact characteristics and levels of protein aggregates that lead to an enhanced immune response are unclear and may differ among proteins, it is not possible to predict, a priori, the in vivo effects of different sizes, types or quantities of aggregates for therapeutic protein products. In such situations, careful analysis of the relationship between clinical performance and the presence of protein aggregates in relevant clinical trial material may help in the design of suitable control strategies that ensure product quality. However, the validity and utility such correlations are only optimized when the full spectrum of protein aggregate species are thoroughly characterized by multiple and orthogonal techniques.

We previously found that EGF (epidermal growth factor) increases the EGFR (EGF receptor) kinase-binding affinity towards the major tyrosine phosphorylation sites in downstream adaptor proteins such as Gab1 (Grb2-associated binding protein 1) and Shc [Src homology 2 (SH2) domain and collagen containing protein], but not that towards EGFR autophosphorylation sites [Fan, Wong, Deb and Johnson (2004) J. Biol. Chem. 279, 38143–38150]. EGFR activation can also result in transphosphorylation of tyrosine resides in the C-terminal region of the related receptors ErbB2, ErbB3 and ErbB4 in heterodimers which are formed upon ligand stimulation. In the present study, we investigated the specificity of EGFR kinase by comparing the steady state kinetic parameters for peptides derived from all four ErbBs in the absence or presence of EGF. Our results demonstrated that (i) EGFR kinase can efficiently phosphorylate a broad range of diverse peptide sequences representing ErbB sites; (ii) certain ErbB2, ErbB3 and ErbB4 sites had higher specificity constants than any EGFR sequence and (iii) EGF stimulation consistently increases the kcat approx. 5-fold, but does not significantly alter the Km for any ErbB peptides. Furthermore, peptides containing lysine at position −2 or −3 N-terminal to the target tyrosine were found to be poor EGFR kinase substrates, and substitution of these lysines with glutamine decreased the Km and increased the kcat for these substrates. We conclude that EGFR kinase-mediated ErbB transphosphorylations are mostly controlled at the level of oligomerization, and not by a preference of the EGFR kinase for phosphorylation sites in any particular ErbB. The results also demonstrated that, unlike phosphorylation sites in select downstream targets, EGF does not regulate the recognition of phosphorylation sites in the C-terminal region of any of the ErbBs.

Current antiestrogen therapy for breast cancer is limited by the mixed estrogenic and antiestrogenic activity of selective estrogen receptor modulators. Here we show that the function of zinc fingers in the estrogen receptor DNA-binding domain (DBD) is susceptible to chemical inhibition by electrophilic disulfide benzamide and benzisothiazolone derivatives, which selectively block binding of the estrogen receptor to its responsive element and subsequent transcription. These compounds also significantly inhibit estrogen-stimulated cell proliferation, markedly reduce tumor mass in nude mice bearing human MCF-7 breast cancer xenografts, and interfere with cell-cycle and apoptosis regulatory gene expression. Functional assays and computational analysis support a molecular mechanism whereby electrophilic agents preferentially disrupt the vulnerable C-terminal zinc finger, thus suppressing estrogen receptor-mediated breast carcinoma progression. Our results provide the proof of principle for a new strategy to inhibit breast cancer at the level of DNA binding, rather than the classical antagonism of estrogen binding.

Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal "cortactin repeats". Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.

Crystal structures of the tryptophan synthase α2β2complex show binding sites in the β subunit for pyridoxal phosphate, for substrates, and for a monovalent cation. Cations, including guanidine at low concentration, activate tryptophan synthase. Molecular modeling indicates that guanidine could bind to the cation site in the β subunit. Our results demonstrate that the conformational states of the tryptophan synthase α2β2 complex are regulated by monovalentcations, chaotropic agents, pH, α subunit ligands, and temperature.

Compared with the activation of dihydrofolate reductase (DHFR) by protein denaturants and inorganic salts, activation of the enzyme by thiol modification is relatively slow. Thus it is an ideal system for kinetic study of the activation mechanism. We describe here a kinetic study of the activation of DHFRs from chicken liver and Chinese hamster ovary by p-hydroxymercuribenzoate (p-HMB). The conformational changes in the enzyme molecule that result from the modification were monitored by measuring fluorescence enhancement due to the binding of 2-p-toluidinylnaphthalene-6-sulphonate (TNS), and by monitoring changes in the intrinsic fluorescence of the enzyme. Both activation and the conformational change probed by TNS followed pseudo-first-order kinetics, and the rate constants obtained are in good agreement with each other. The change in intrinsic fluorescence is a biphasic process. The rate of the fast phase, which may reflect a change in the microenvironment of Trp-24 at the active site, coincides with the rate of activation and the conformational change probed by TNS. The rate of the slow phase, which reflects a global conformational change, is about one order of magnitude lower than that of activation. The results indicate that the activation of DHFR by p-HMB is due to modification-induced conformational changes at its active site, rather than the modification of the thiol group itself, which is almost complete within the dead-time of the experiment. This study provides kinetic evidence for the proposal that flexibility at the active site is essential for full expression of catalytic activity.

The unfolding of creatine kinase in various concentrations of guanidine hydrochloride of increasing concentrations has been investigated by combination of size-ex clusion chromatography (SEC) with other methods. There are two peaks in the profiles of SEC in GuHCl at moderate concentrations, showing that unfolding of creatine kinase goes through dimeric and monomeric intermediates with increasing guanidine hydrochloride concentrations. Both intermediates have relatively compact structure and retain considerable ordered structure.

Intersubunit cross-linked creatine kinase (CK) has been prepared with the cross-linking reagent dithiobis(succinimidyl propionate) (DTSP). Unfolding of cross-linked CK during denaturation by guanidine hydrochloride (GuHC1), as monitored by intrinsic fluorescence, circular dichroism and fluorescence of the hydrophobic probe, 1-aniline-naphthalene-8-sulfonate (ANS), occurs in two stages with increasing GuHC1 concentration. The process is similar to that of the unmodified enzyme, but in the second stage, conformational changes of the cross-linked enzyme need higher concentration of GuHC1, suggesting that there is a stable intermediate during its unfolding transition and the intermediate is stabilized by intersubunit cross-linkage.

Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors. The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell. With repeated cycles of freezing and thawing as a first step, the purification of the recombinant ch-DHFR to homogeneity requires only one further step, gel filtration on a Sephadex G-75 column with 85-90% enzyme recovery, two to three times higher than that obtained with the commonly used affinity chromatography on a methotrexate-Sepharose column. The purified enzyme migrates as a single protein band on SDS-polyacrylamide gel electrophoresis with approximate mass of 23 kDa, in accord with that calculated from the DNA sequence. The initiation methionine residue at the N-terminus of the enzyme is completely removed by E. coli methionine aminopeptidase as judged by amino-terminal analysis. The steady-state kinetic parameters, dissociation constants for binary complexes of dihydrofolate, NADPH, and methotrexate with ch-DHFR, and the inhibitor constant of methotrexate have also been determined. The enzyme is activated about 4-fold in 3 M urea and about 2.5-fold in 0.5 M guanidine hydrochloride.

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hydrochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteolysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conformational change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0. 75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at the active site first, until to a critical concentration of the denaturant the overall conformation change of the molecule may occur cooperatively. Activation of DHFR in low GuHCl concentration is due to the subtle conformational change at the active site of the enzyme. Although the conformational change at the active site decreases the affinity of the enzyme for the substrates, it accelerates the velocity of the limiting step in the catalytic reaction, i.e. the velocity of the dissociation of products from the ternary complex.

The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3-fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed.

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R g value of about 17.3 A in 8 M urea. The reduced ribonuclease A is more expanded, its R g value is about 20 A in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.

The denaturation of dimeric creatine kinase (CK) induced by urea and guanidine hydrochloride (GuHCl) has been studied by small angle X-ray scattering (SAXS), which is a direct way to measure the changes in the overall dimensions of a protein molecule. The radii of gyration (Rg) of CK are 29+/-0.4 angstroms in the native state and 46+/-1.5 angstroms in the unfolded state in either 8 M urea or 3 M GuHCl. The transition curves of urea denaturation derived from the Rg values and the zero angle intensity (I(0)) are similar to that from intrinsic fluorescence, indicating that the changes in the molecular shape, the tertiary structure and the dissociation of the subunits proceed simultaneously. In the case of GuHCl-induced denaturation, the dramatic increases both in Rg and in I(0) in 0.3-0.5 M GuHCl suggest clearly that soluble aggregates form at low GuHCl concentrations. The aggregates dissociate and the molecule unfolds at higher GuHCl concentrations. The results suggest that the mechanisms of CK denaturation in urea and in GuHCl are somewhat different and the intermediate in GuHCl denaturation can much more easily form soluble aggregates.