RESUMO
Asn deamidation was monitored in Ala-based octadecapeptides of varying alpha-helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala --> Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed-phase high-performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of alpha-helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.
Assuntos
Asparagina , Peptídeos/química , Estrutura Secundária de Proteína , Alanina , Sequência de Aminoácidos , Glicina , Cinética , Dados de Sequência Molecular , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The effect of pH and urea on the conformation of recombinant human megakaryocyte growth and development factor (rHuMGDF) was determined by circular dichroism, intrinsic fluorescence spectroscopy, and equilibrium ultracentrifugation. The conformation of rHuMGDF was dependent on pH and urea concentration. Multiple folding forms were evidenced by multiple pH-induced transitions and urea-induced equilibrium transitions that deviated from a simple two-state process. In neutral to alkaline pH, rHuMGDF exists as a monomer, but an acid-induced conformational state self-associates to form a soluble aggregate. A folding intermediate(s) was observed with a more stable secondary structure than tertiary structure and was dependent on the pH of the urea-induced denaturation. The differences in the stabilities of the folding states were most distinct in the pH range of 4.5 to 6.5. The presence of intermediates in the folding pathway of rHuMGDF are similar to findings of previous studies of related growth factors that share a common three-dimensional structure.
Assuntos
Trombopoetina/química , Dicroísmo Circular , Citocinas/química , Humanos , Concentração de Íons de Hidrogênio , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Raios Ultravioleta , UreiaRESUMO
PURPOSE: Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range. METHODS: Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry. RESULTS: The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDE This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG). CONCLUSIONS: PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhanced in-vitro shelf-life stability.
Assuntos
Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Trombopoetina/química , Trombopoetina/metabolismo , Alquilação , Antibacterianos , Cromatografia Líquida de Alta Pressão , Dicetopiperazinas , Armazenamento de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Piperazinas , Polietilenoglicóis/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Trombopoetina/análiseRESUMO
The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.
Assuntos
Insulina/análogos & derivados , Regulação Alostérica , Feminino , Humanos , Insulina/química , Insulina/metabolismo , Insulina Lispro , Ligantes , Ligação Proteica , Conformação ProteicaRESUMO
The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40-60% of expected synthetase activity along with 50-80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step. Further purification to homogeneity was achieved by a single anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation. Information obtained from refolding studies using gel-filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e. 65-75% of the expected activity) and moderately stable fungal synthetase/hydroxylase.
Assuntos
Acremonium/enzimologia , Escherichia coli/genética , Transferases Intramoleculares , Isomerases/química , Isomerases/genética , Proteínas de Ligação às Penicilinas , Dissulfetos/química , Estabilidade Enzimática , Escherichia coli/química , Escherichia coli/enzimologia , Isomerases/metabolismo , Modelos Químicos , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/químicaRESUMO
We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Hexosiltransferases , Resistência a Meticilina/fisiologia , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Peptidil Transferases , Staphylococcus aureus/metabolismo , Acilação , Antibacterianos/química , Proteínas de Transporte/química , Cefalosporinas/farmacologia , Compostos Cromogênicos/química , Cinética , Lactamas , Espectrometria de Massas , Muramilpentapeptídeo Carboxipeptidase/química , Penicilina V/farmacologia , Proteínas de Ligação às Penicilinas , Penicilinas/farmacologia , Ligação Proteica , Tiazóis/farmacologia , Ureia/química , beta-Lactamas/farmacologiaRESUMO
PURPOSE: The liquid stability of rhG-CSF was investigated after polyethylene glycol (PEG) with an average molecular weight of 6000 daltons was covalently attached to the N-terminal methionine residue. METHODS: The conjugation methods chosen for modifying the N-terminal residue were alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography. The physical characterization methods of SDS-PAGE, endoproteinase peptide mapping, circular dichroism and in-vivo bioassay were used to test for differences between the PEG-rhG-CSF molecules. RESULTS: Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temperatures, of these conjugated molecules indicated that the primary pathway of degradation was aggregation. Conjugation through alkylation offered the distinct advantage of decreasing, by approximately 5 times, the amount of aggregation present as compared to acylation. CONCLUSIONS: We suggest, that the increased stability observed for the molecules utilizing the alkylation conjugation method may be due to the preservation of charge on the alpha amino group of rhG-CSF.
Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/análogos & derivados , Polietilenoglicóis , Proteínas RecombinantesRESUMO
We report the results of a stopped-flow kinetic evaluation of the folding of human growth hormone (hGH). The results are compared with those obtained for a disulfide-modified analog in which the four cysteine residues have been reduced and alkylated to form tetra-S-carbamidomethylated hGH in order to elucidate the role of disulfide bonds in the folding reaction. Multiple detection techniques were applied to monitor both refolding and unfolding processes initiated by guanidine hydrochloride concentration jumps. Using far-UV circular dichroism (CD) detection to monitor folding of hGH, we find that 70% of the secondary structure forms in a burst phase occurring within the stopped-flow dead time. Two slower phases were identified in the observable portion of the CD signal. Multiple kinetic phases were resolved when folding was monitored by intrinsic tryptophan fluorescence or near-UV absorbance as probes of tertiary structure, and the number of time constants required to fit the data depended on the hGH concentration and nature of the denaturant jump. The associated amplitudes also displayed strong dependence on the final denaturant concentration. Results obtained from the tetra-S-carbamidomethylated hGH studies demonstrate that the folding reactions of hGH are remarkably similar in the presence and absence of the disulfide bonds. Disulfide bond reduction in hGH is proposed to affect folding primarily by increasing the population of self-associated intermediate states in the folding pathway.
Assuntos
Hormônio do Crescimento/química , Animais , Bovinos , Dicroísmo Circular , Dissulfetos/química , Hormônio do Crescimento/análogos & derivados , Guanidina , Guanidinas/farmacologia , Humanos , Técnicas In Vitro , Cinética , Estrutura Molecular , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B9-B20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (ValB18 and TyrB16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.
Assuntos
Amidas/metabolismo , Insulina/química , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , Sítio Alostérico , Deutério , Humanos , Substâncias Macromoleculares , Fenol , Fenóis/farmacologia , PrótonsRESUMO
The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).
Assuntos
Fosfolipases A/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Catálise , Sequência Consenso , Citosol/enzimologia , Humanos , Hidrólise , Lisofosfolipase/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfolipases A/química , Fosfolipases A2 , Homologia de Sequência de AminoácidosRESUMO
Amyloid beta peptide (A beta), the major protein constituent of senile plaques in patients with Alzheimer's disease, is believed to facilitate the progressive neurodegeneration that occurs in the latter stages of this disease. Early attempts to characterize the structure-activity relationship of A beta toxicity in vitro were compromised by the inability to reproducibly elicit A beta-dependent toxicity across different lots of chemically equivalent peptides. In this study we used CD spectroscopy to demonstrate that A beta secondary structure is an important determinant of A beta toxicity. Solubilized A beta was maximally toxic when the peptide adopted a beta-sheet conformation. Three of the four A beta lots tested had a random coil conformation and were weakly toxic or inactive, whereas the single A beta lot exhibiting toxic activity at low peptide concentrations had significant beta-sheet structure. Incubation of the weakly toxic A beta lots in aqueous stock solutions for several days before use induced a time-dependent conformational transition from random coil to beta-sheet and increased A beta toxicity in three different toxicity assays. Furthermore, the secondary structure of preincubated A beta was dependent upon peptide concentration and pH, so that beta-sheet structures were attenuated when peptide solutions were diluted or buffered at neutral and basic pH. Our data could explain some of the variable toxic activity that has been associated with A beta in the past and provide additional support for the hypothesis that A beta can have a causal role in the molecular neuropathology of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/química , Neurônios/efeitos dos fármacos , Estrutura Secundária de Proteína , Peptídeos beta-Amiloides/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Neurônios/química , Neurônios/citologia , RatosRESUMO
The equilibrium denaturation of human insulin in a monomer-inducing solvent and of two monomeric insulin analogs, lysB28proB29 insulin and aspB10desB28-30 insulin, was reexamined [Brems, D. N., Brown, P. L., Heckenlaible, L. A., & Frank, B. H. (1990) Biochemistry 29,9289-9293] by circular dichroism (CD) at additional wavelengths in the near-UV region. Previous denaturation studies were limited by the solubility of guanidine hydrochloride being only slightly greater than the level of denaturant required to fully unfold human insulin; therefore, only a few data points were available for construction of the post-transitional baseline. In the present study, we report the use of an unfolded mimic created by enzymatic digestion of insulin to confirm the slope of the post-transitional baseline. Evidence for equilibrium unfolding intermediates for each of these insulins was indicated by noncoincidence of the denaturation transitions as monitored by tyrosine and helical-dependent CD bands (270 and 224 nm, respectively). Additional evidence for intermediates through multiphasic denaturation transitions was obtained at a wavelength likely related to disulfide conformation, 251 nm. The results suggest that for each of the insulins, at least two intermediates are significantly populated. An unfolding model is proposed in which the conformation of the least stable intermediate is slightly unfolded only in the C-terminal segment of the B chain. A second more stable intermediate retains minimal secondary structure while containing localized structure proximal to one or more of the disulfide groups. The presence of equilibrium intermediates has important implications for the folding pathway of insulin, in pharmaceutical applications such as formulation stability, and for conformational transitions that accompany receptor binding.
Assuntos
Insulina/química , Animais , Dicroísmo Circular , Guanidina , Guanidinas , Humanos , Insulina/análogos & derivados , Masculino , Desnaturação Proteica , Dobramento de Proteína , Ratos , Ratos Sprague-Dawley , Espectrofotometria UltravioletaRESUMO
The effect of pH on the conformational stability of insulin was studied. Surprisingly, the Gibbs free energy of unfolding increased approximately 30% by acidification. pH titration of insulin's conformational stability is described by a transition involving a single proton with an apparent pK(a) of 7.0. The acid stabilization of insulin's conformation was attributed to the protonation of histidine at position 5 on the B-chain (HB5) as determined by 1H-NMR of the histidines, selective amino acid alteration, and enthalpies of ionization. Further acidification (at least to pH 2) does not decrease the free energy of unfolding. A conformational change in the tertiary structure, as indicated by the near-UV circular dichroism spectrum, accompanies this change in stability. We propose that this acid stabilization of insulin is physiologically important in maintaining insulin stability in the acid environment of the secretory/storage granules of the beta-cell of the pancreatic islets of Langerhans.
Assuntos
Insulina/química , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , Estabilidade de Medicamentos , Histidina/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Proteica , Prótons , TermodinâmicaRESUMO
It has been previously shown, by equilibrium denaturation, that human growth hormone (hGH) folds by a cooperative two-state process. This is in contrast to the folding pathways of other nonhuman growth hormones that contain stable monomeric and multimeric equilibrium intermediates. We have reinvestigated the equilibrium denaturation of hGH at higher protein concentrations and found smooth transitions from the native to denatured state, but the calculated free energy for unfolding, delta G, decreases with increasing protein concentration. The effect of protein concentration on the delta G of unfolding is due to the presence of folding intermediates that have a tendency to self-associate. A correlation was found between the equilibrium denaturation data and the observation of precipitation that occurs upon refolding, suggesting that the presence of self-associated folding intermediates leads to precipitation. Direct evidence for the existence of a soluble, associated intermediate was obtained by dynamic light scattering (DLS) and equilibrium analytical ultracentrifugation. Peptide fragments from the third helix of either hGH or bovine growth hormone (bGH) were capable of inhibiting the formation of this aggregated species and prevent precipitation during refolding. The data show that the folding pathway of hGH is similar to that of nonhuman growth hormones except for differences in the tendency for intermediates to self-associate. These findings are relevant to the design and interpretation of equilibrium folding experiments, and may be important to understanding mechanistic details of protein folding and aggregation in vivo.
Assuntos
Hormônio do Crescimento/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Cromatografia Líquida de Alta Pressão , Hormônio do Crescimento/metabolismo , Guanidina , Guanidinas/farmacologia , Humanos , Cinética , Luz , Peso Molecular , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento de RadiaçãoRESUMO
Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.
Assuntos
Insulina/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Cistina , Estabilidade de Medicamentos , Insulina/análogos & derivados , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Polímeros , Conformação Proteica , Desnaturação Proteica , Temperatura , TermodinâmicaRESUMO
The importance of ProB28 and LysB29 on the self-association of insulin was established by systematically truncating the C terminus of the B chain. The relationship between structure and association was further explored by making numerous amino acid replacements at B28 and B29. Association was studied by circular dichroism, size-exclusion chromatography and ultracentrifugation. Our results show that the location of a prolyl residue at B28 is critical for high-affinity self-association. Removal of ProB28 in a series of C-terminal truncated insulins, or amino acid replacement of ProB28, greatly reduced association. The largest disruption to association was achieved by replacing LysB29 with Pro and varying the amino acid at B28. Several of the analogs were predominantly monomers in solutions up to 3 mg/ml. These amino acid substitutions decreased association by primarily disrupting the formation of dimers. Such amino acid substitutions also substantially reduced the Zn-induced insulin hexamer formation. The formation of monomeric insulins through amino acid replacements was accompanied by conformational changes that may be the cause for decreased association. It is demonstrated that self-association of insulin can be drastically altered by substitution of one or two key amino acids.
Assuntos
Insulina/química , Conformação Proteica , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Humanos , Insulina/análogos & derivados , Lisina , Modelos Moleculares , Dados de Sequência Molecular , Prolina , Dobramento de Proteína , Ultracentrifugação , ZincoRESUMO
To determine the conformational properties of the C-terminal region of the insulin B-chain relative to the helical core of the molecule, we have investigated the fluorescence properties of an insulin analog in which amino acids B28 and B29 have been substituted with a tryptophan and proline residue respectively, ([WB28,PB29]insulin). The biological properties and far-UV circular dichroism (CD) spectrum of the molecule indicate that the conformation is similar to that of native human insulin. Guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation of the analog as monitored by CD intensity at 224 nm indicates a single cooperative transition with a midpoint of 4.9 M GdnHCl. In contrast, when the equilibrium denaturation is observed by steady-state fluorescence emission intensity at 350 nm, two distinct transitions are observed. The first transition accounts for 60% of the observed signal and has a midpoint of 1.5 M GdnHCl. The second transition roughly parallels that observed by CD measurements with an approximate midpoint of 4.5 M GdnHCl. The near-UV CD spectrum, size-exclusion, and ultracentrifugation properties of [WB28,PB29]insulin indicate that this analog does not self-associate in a concentration-dependent manner as does human insulin. Thus, the observed fluorescence changes must be due to specific conformational transitions which occur upon unfolding of the insulin monomer with the product of the first transition representing a stable folding intermediate of this molecule.
Assuntos
Insulina/análogos & derivados , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Dicroísmo Circular , Guanidina , Guanidinas , Insulina/síntese química , Insulina/química , Insulina/farmacologia , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Ratos , Ratos Endogâmicos , Espectrometria de FluorescênciaRESUMO
The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.
Assuntos
Insulina , Animais , Dicroísmo Circular , Reagentes de Ligações Cruzadas , Deutério/química , Guanidina , Guanidinas/farmacologia , Técnicas In Vitro , Insulina/química , Movimento (Física) , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , SuínosRESUMO
The guanidine hydrochloride induced equilibrium denaturation of insulin and proinsulin was studied by using near- and far-ultraviolet (UV) circular dichroism (CD). The denaturation transition of insulin is reversible, cooperative, symmetrical, and the same whether detected by near- or far-UV CD. These results are consistent with a two-state denaturation process without any appreciable equilibrium intermediates. Analysis of the insulin denaturation data yields a Gibbs free energy of unfolding of 4.5 +/- 0.5 kcal/mol. Denaturation of proinsulin detected by near-UV CD appears to be the same as for insulin, but if detected by far-UV CD appears different. The far-UV CD results demonstrate a multiphasic transition with the connecting peptide portion unfolding at lower concentrations of denaturant. Similar studies with the isolated C-peptide show that its conformation and susceptibility to denaturation are independent of the rest of the proinsulin molecule. After the proinsulin denaturation results were adjusted for the connecting peptide contribution, a denaturation transition identical with that of insulin was obtained. These results show that for proinsulin, the connecting peptide segment is not a random coil; it is an autonomous folding unit, and the portion corresponding to insulin is identical with insulin in terms of conformational stability.
Assuntos
Insulina/metabolismo , Proinsulina/metabolismo , Dicroísmo Circular , Etanol/farmacologia , Guanidina , Guanidinas/farmacologia , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , TermodinâmicaRESUMO
The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this.