ABSTRACT
BACKGROUND AND PURPOSE: FM19G11 up-regulates mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α) and PI3K/Akt pathways, which are involved in endothelial function. We evaluated the effects of FM19G11 on defective endothelial vasodilatation in arteries from rats and humans and investigated the mechanisms involved. EXPERIMENTAL APPROACH: Effects of chronic in vivo administration of FM19G11 on aortic endothelial vasodilatation were evaluated together with ex vivo treatment in aortic and mesenteric arteries from control and insulin-resistant rats (IRR). Its effects on vasodilator responses of penile arteries (HPRAs) and corpus cavernosum (HCC) from men with vasculogenic erectile dysfunction (ED) (model of human endothelial dysfunction) were also evaluated. Vascular expression of phosphorylated-endothelial NOS (p-eNOS), phosphorylated-Akt (p-Akt) and HIF-1α was determined by immunodetection and cGMP by elisa. KEY RESULTS: Chronic administration of FM19G11 reversed the impaired endothelial vasodilatation in IRR. Ex vivo treatment with FM19G11 also significantly improved endothelium-dependent vasodilatation in aorta and mesenteric arteries from IRR. These effects were accompanied by the restoration of p-eNOS and cGMP levels in IRR aorta and were prevented by either NOS or PI3K inhibition. p-Akt and p-eNOS contents were increased by FM19G11 in aortic endothelium of IRR. FM19G11-induced restoration of endothelial vasodilatation was unaffected by mTOR/HIF-1α inhibitors. FM19G11 also restored endothelial vasodilatation in HPRA and HCC from ED patients. CONCLUSIONS AND IMPLICATIONS: Stimulation of the PI3K/Akt/eNOS pathway by FM19G11 alleviates impaired NO-mediated endothelial vasodilatation in rat and human arteries independently of mTOR/HIF-1α activation. This pharmacological strategy could be beneficial for managing pathological conditions associated with endothelial dysfunction, such as ED.
Subject(s)
Arteries/drug effects , Benzamides/pharmacology , Endothelium, Vascular/drug effects , Nitric Oxide Synthase Type III/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Adult , Aged , Arteries/metabolism , Benzamides/administration & dosage , Endothelium, Vascular/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Middle Aged , TOR Serine-Threonine Kinases/metabolism , Young AdultABSTRACT
A novel magnetobiosensing approach for the rapid and simultaneous detection of two breast cancer-related miRs (miR-21 and miR-205) is reported. It involves the use of antimiR-21 and antimiR-205 specific probes, chitin-modified magnetic beads (Chitin-MBs), the p19 viral protein as capture bioreceptor and amperometric detection with the H2O2/hydroquinone (HQ) system at dual screen-printed carbon electrodes (SPdCEs). The use of SPdCEs allows the simultaneous independent amperometric readout for each target miR to be measured. The magnetosensor exhibited sensitive and selective detection with dynamic ranges from 2.0 to 10.0nM and detection limits of 0.6nM (6fmol) for both target miRs without any amplification step in less than 2h. The usefulness of the approach was evaluated by detecting the endogenous levels of both target miRs in total RNA (RNAt) extracted from metastatic breast cancer cell lines and human tissues.
Subject(s)
Biosensing Techniques/instrumentation , Breast Neoplasms/genetics , MicroRNAs/analysis , Breast/metabolism , Electrochemical Techniques/instrumentation , Equipment Design , Female , Humans , Limit of Detection , Magnetics/instrumentation , MicroRNAs/genetics , Viral Core Proteins/chemistryABSTRACT
1. Predators often prey on individuals that are sick or otherwise weakened. Although previous studies have shown higher abundance of parasites in prey, whether prey have elevated loads of micro-organisms remains to be determined. 2. We quantified the abundance of bacteria and fungi on feathers of woodpigeons Columba palumbus L., jays Garrulus glandarius L. and blackbirds Turdus merula L. that either fell prey to goshawks Accipiter gentilis L. or were not depredated. 3. We found an almost three-fold increase in bacterial load of prey compared with non-prey, while there was no significant difference between prey and non-prey in level of fungal infection of the plumage. 4. The results were not confounded by differences in size or mass of feathers, date of collection of feathers, or date of analysis of feathers for micro-organisms. 5. These findings suggest a previously unknown contribution of bacteria to risk of predation, with important implications for behaviour, population ecology and community ecology.
Subject(s)
Bacteria/isolation & purification , Feathers/microbiology , Food Chain , Fungi/isolation & purification , Hawks/physiology , Animals , Bacterial Load , Colony Count, Microbial , Columbidae/microbiology , Denmark , Predatory Behavior , Songbirds/microbiologyABSTRACT
BACKGROUND. In 1997, The Fourth International Workshop-Conference on Gestational Diabetes Mellitus (GDM) recommended the use of Carpenter and Coustan diagnostic criteria instead of those of Sullivan and Mahan used until then. The objective of this work was to study the incidence of GDM in the southeast Madrid area by applying both the classical and new criteria, as well as to determine whether patients diagnosed of GDM by the new criteria present a higher materno-fetal morbidity than treated GDM patients or non diabetic pregnant patients.Methods. Review of a cohort of 1,293 pregnant women from September 1998 to August 1999 who were screened for GDM. The annual cumulated incidence of GDM was estudied by using both diagnostic criteria and materno-fetal evolution of four groups of patients classified according to glucose intolerance degree. RESULTS. The annual cumulated incidence of GDM was 4.8% and 7.3% using the classical criteria and the new criteria, respectively. GDM patients fulfilling only the new criteria and not treated as such had children with a significantly higher birth-weight than the other groups, and a trend towards a higher percentage of macrosomies, instrumental deliveries and cesarean sections in such group was observed, although they were not statistically significant. CONCLUSIONS. The incidence of GDM in the southeast Madrid area was similar to that among other Caucasian populations and increased by 52% when the new diagnostic criteria were used. It is our opinion that a study with a larger number of patients should be conducted before the new diagnostic criteria are applied in our country.
ABSTRACT
Objetivo. En 1997, la Cuarta Conferencia Internacional sobre diabetes mellitus gestacional (DMG) recomendó la aplicación de los criterios diagnósticos de Carpenter y Coustan en lugar de los criterios de OSullivan y Mahan utilizados hasta entonces. El objetivo de este trabajo es estudiar la incidencia de DMG en el área suroeste de Madrid aplicando los criterios clásicos y nuevos, así como determinar si las pacientes diagnosticadas de DMG por los nuevos criterios presentan una mayor morbilidad maternofetal que las DMG tratadas o las gestantes no diabéticas. Metodología. Revisión de una cohorte de 1 .2 9 3 gestantes durante el período de septiembre de 1998-agosto de 1999 a las que se les realizó despistaje de DMG. Se estudia la incidencia acumulada anual de DMG utilizando ambos criterios diagnósticos y la evolución maternofetal de cuatro grupos de pacientes clasificadas según el grado de intolerancia a la glucosa. Resultados. La incidencia acumulada anual de DMG fue del 4 ,8 % utilizando los criterios clásicos, y del 7,3% aplicando los nuevos criterios. Las pacientes con DMG que sólo cumplían los nuevos criterios y no fueron tratadas como tales tuvieron hijos con un peso sensiblemente mayor que el resto de grupos, observándose una tendencia a un mayor porcentaje de macrosomías, partos instrumentales y cesáreas en dicho grupo, aunque sin alcanzar significación en la estadística. Conclusiones. La incidencia de DMG en el área suroeste de Madrid es similar a la de otras poblaciones caucásicas, y se incrementa en un 5 2 % al aplicar los nuevos criterios diagnósticos. Creemos necesaria la realización de un estudio con mayor número de pacientes antes de aplicar los nuevos criterios diagnósticos en nuestro país (AU)
Background. In 1997, The Fourth International Workshop-Conference on Gestational Diabetes Mellitus (GDM) recommended the use of Carpenter and Coustan diagnostic criteria instead of those of Sullivan and Mahan used until then. The objective of this work was to study the incidence of GDM in the southeast Madrid area by applying both the classical and new criteria, as well as to determine whether patients diagnosed of GDM by the new criteria present a higher materno-fetal morbidity than treated GDM patients or non diabetic pregnant patients. Methods. Review of a cohort of 1,293 pregnant women from September 1998 to August 1999 who were screened for GDM. The annual cumulated incidence of GDM was estudied by using both diagnostic criteria and materno-fetal evolution of four groups of patients classified according to glucose intolerance degree. Results. The annual cumulated incidence of GDM was 4 .8 % and 7 .3 % using the classical criteria and the new criteria, respectively. GDM patients fulfilling only the new criteria and not treated as such had children with a significantly higher birth- weight than the other groups, and a trend towards a higher percentage of macrosomies, instrumental deliveries and cesarean sections in such group was observed, although they were not statistically significant. Conclusions. The incidence of GDM in the southeast Madrid area was similar to that among other Caucasian populations and increased by 5 2 % when the new diagnostic criteria were used. It is our opinion that a study with a larger number of patients should be conducted before the new diagnostic criteria are applied in our country (AU)
Subject(s)
Female , Humans , Pregnancy , Diabetes, Gestational/epidemiology , Hyperglycemia/epidemiology , Pregnancy Complications/epidemiology , Rural Population/statistics & numerical data , Glucose Tolerance Test , Mass Screening/methods , Case-Control StudiesABSTRACT
El síndrome de Wolfram es un cuadro neurodegenerativo hereditario con transmisión autosómica recesiva, caracterizado por la asociación de diabetes mellitus de inicio en la juventud y atrofia óptica, y presenta con frecuencia otras manifestaciones clínicas como diabetes insípida central y sordera neurosensorial. En el presente artículo se expone el caso índice de una nueva familia con síndrome de Wolfram, cuyo principal hallazgo de interés fue la presencia, en la resonancia magnética, de alteraciones morfológicas encefálicas características y que han sido descritas recientemente en un 18 por ciento de los pacientes con este síndrome (AU)
Subject(s)
Adult , Male , Humans , Wolfram Syndrome/diagnosis , Diabetes Mellitus, Type 1/complications , Telencephalon/abnormalitiesABSTRACT
The signal transduction protein CheY displays an alpha/beta-parallel polypeptide folding, including a highly unstable helix alpha4 and a strongly charged active site. Helix alpha4 has been shown to adopt various positions and conformations in different crystal structures, suggesting that it is a mobile segment. Furthermore, the instability of this helix is believed to have functional significance because it is involved in protein-protein contacts with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ. The active site of CheY comprises a cluster of three aspartic acid residues and a lysine residue, all of which participate in the binding of the Mg(2+) needed for the protein activation. Two steps were followed to study the activation mechanism of CheY upon phosphorylation: first, we independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix alpha 4, both to increase its level of stability and to improve its packing against the protein core. The structural and thermodynamic analysis of these mutant proteins provides further evidence of the connection between the active-site area and helix alpha 4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface.
Subject(s)
Escherichia coli/chemistry , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Signal Transduction , Amino Acid Substitution/genetics , Apoproteins/chemistry , Apoproteins/genetics , Apoproteins/metabolism , Aspartic Acid/genetics , Aspartic Acid/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli Proteins , Histidine Kinase , Magnesium/metabolism , Membrane Proteins/genetics , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Point Mutation/genetics , Protein Binding , Protein Denaturation/drug effects , Protein Structure, Secondary/drug effects , Structure-Activity Relationship , Thermodynamics , Urea/pharmacology , Water/metabolismABSTRACT
A number of reports have identified phosphatidylinositol 3-kinase as a downstream effector of Ras in various cellular settings, in contrast to others supporting the notion that phosphatidylinositol 3-kinase acts upstream of Ras. Here, we used Xenopus oocytes, a model of Ras-mediated cell cycle progression (G2/M transition) to analyze the contribution of phosphatidylinositol 3-kinase to insulin/Ras-dependent signaling pathways leading to germinal vesicle breakdown and to ascertain whether phosphatidylinositol 3-kinase acts upstream or downstream of Ras in those signaling pathways. We analyzed the process of meiotic maturation induced by progesterone, insulin or micro-injected oncogenic Ras (Lys12) proteins in the presence and absence of specific inhibitors of phosphatidylinositol 3-kinase activity. As expected, the progesterone-induced maturation was independent of phosphatidylinositol 3-kinase since similar rates of germinal vesicle breakdown were produced by the hormone in the presence and absence of wortmannin and LY294002. In contrast, insulin-induced germinal vesicle breakdown was completely blocked by pre-incubation with the inhibitors prior to insulin treatment. Interestingly, similar rates of germinal vesicle breakdown were obtained in Ras (Lys12)-injected oocytes, independently of whether or not they had been pre-treated with phosphatidylinositol 3-kinase inhibitors. The effect of wortmannin or LY294002 on MAPK and Akt activation by progesterone, insulin or Ras was also analyzed. Whereas insulin activated those kinases in a phosphatidylinositol 3-kinase-dependent manner, progesterone and Ras were able to activate those kinases in the absence of phosphatidylinositol 3-kinase activity. Since Ras is a necessary and sufficient downstream component of insulin signaling pathways leading to germinal vesicle breakdown, these observations demonstrate that phosphatidylinositol 3-kinase is not a downstream effector of Ras in insulin/Ras-dependent signaling pathways leading to entry into the M phase in Xenopus oocytes.
Subject(s)
Oocytes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , ras Proteins/metabolism , Androstadienes/pharmacology , Animals , Cell Nucleus/metabolism , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Insulin Antagonists/pharmacology , Meiosis , Morpholines/pharmacology , Oocytes/cytology , Signal Transduction/drug effects , Wortmannin , XenopusABSTRACT
The crystal structures of two double mutants (F14N/V21T and F14N/V86T) of the signal transduction protein CheY have been determined to a resolution of 2.4 and 2.2 A, respectively. The structures were solved by molecular replacement and refined to final R values of 18.4 and 19.2%, respectively. Together with urea-denaturation experiments the structures have been used to analyse the effects of mutations where hydrophobic residues are replaced by residues capable of establishing hydrogen bonds. The large increase in stabilization (-12.1 kJ mol-1) of the mutation Phe14Asn arises from two factors: a reverse hydrophobic effect and the formation of a good N-cap at alpha-helix 1. In addition, a forward-backward hydrogen-bonding pattern, resembling an N-capping box and involving Asn14 and Arg18, has been found. The two Val to Thr mutations at the hydrophobic core have different thermodynamic effects: the mutation Val21Thr does not affect the stability of the protein while the mutation Val86Thr causes a small destabilization of 1.7 kJ mol-1. At site 21 a backward side chain-to-backbone hydrogen bond is formed inside alpha-helix 1 with the carbonyl O atom of the i - 4 residue without movement of the mutated side chain. The destabilizing effect of introducing a polar group in the core is efficiently compensated for by the formation of an extra hydrogen bond. At site 86 the new Ogamma atom escapes from the hydrophobic environment by a chi1 rotation into an adjacent hydrophilic cavity to form a new hydrogen bond. In this case the isosteric Val to Thr substitution is disruptive but the loss in stabilization energy is partly compensated by the formation of a hydrogen bond. The two crystal structures described in this work underline the significance of the hydrogen-bond component to protein stability.
Subject(s)
Bacterial Proteins/chemistry , Chemotaxis , Membrane Proteins/chemistry , Bacterial Proteins/genetics , Hydrogen Bonding , Membrane Proteins/genetics , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Mutation , Protein Conformation , ThermodynamicsABSTRACT
The backbone internal dynamics of the wild-type 129 amino acid alpha/beta parallel protein CheY and its double mutant F14N/P110G are analysed here by the hydrogen-exchange method. The F14N mutation is known to stabilise the protein and to accelerate refolding while P110G is destabilising and accelerates unfolding. We first assigned and characterised the double mutant by nuclear magnetic resonance (NMR), to try and discover any possible conformational change induced by the two mutations. The main difference between the two proteins is a favourable N-capping interaction of the newly introduced Asn14 side-chain at the beginning of the first alpha-helix (alpha-helix A). Second, we have measured the exchange rates in the wild-type and mutant CheY. In the first case the observed protection factors are slightly dispersed around an average value. According to their distribution in the structure, protein stability is highest on one face of the central beta-sheet, in the surroundings of the main hydrophobic core formed by side-chains of residues in beta-strands I, II and III and helices A and E. The mutations in the double mutant protein affect two distinct subdomains differently (from beta-strand I to III and from alpha-helix C to the end). In the second subdomain the number of protected protons is reduced with respect to those in the wild-type. This differential behaviour can be explained by a selective decrease in stability of the second folding subdomain produced by the P110G mutation and the opposite effect in the first subdomain, produced by the F14N mutation. alpha-Helix A, which is involved together with beta-strands I and III in the folding nucleus of CheY, shows the largest protection factors in both proteins.
Subject(s)
Amides/chemistry , Bacterial Proteins/chemistry , Chemotaxis , Escherichia coli/chemistry , Hydrogen , Membrane Proteins/chemistry , Protein Folding , Amino Acid Sequence , Bacterial Proteins/genetics , Escherichia coli/physiology , Escherichia coli Proteins , Magnetic Resonance Spectroscopy , Membrane Proteins/genetics , Methyl-Accepting Chemotaxis Proteins , Molecular Sequence Data , Mutation , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
We present a case of non-Hodgkins lymphoma located in both adrenal glands, with diminished adrenal reserve and fatal evolution with serious metabolic complications, with hypoglycemia, severe lactic acidosis, hyperuricemia, acute renal failure, hepatic affectation and hemogram alterations. Much of these complications can be explained by tumoral lysis syndrome probably prompted by the use of high doses of corticosteroids. Primary adrenal lymphoma is exceptional with only 14 cases described in the literature. In spite of its rarity it should be included in the differential diagnosis of uni or bilateral adrenal masses and an early diagnosis is necessary in order to avoid serious and potentially lethal complications. Percutaneous aspiration biopsy can be a valid method of diagnosis because it can identify specific tumoral antigens. The literature concerning this unusual tumour is reviewed.
Subject(s)
Adrenal Gland Neoplasms/diagnosis , Lymphoma, Non-Hodgkin/diagnosis , Aged , Female , HumansABSTRACT
In this work we study the folding kinetics of Che Y mutants in which the helical propensity of each of its five alpha-helices has been greatly enhanced by local interactions (between residues close in sequence). This constitutes an experimental test on the role of local interactions in protein folding, as well as providing new information on the details of the folding pathway of the protein Che Y. With respect to the first issue, our results show that the enhancement of helical propensities by native-like local interactions in Che Y has the following general effects: (1) the energetics of the whole Che Y folding energy landscape (folded state, intermediate, denatured state and main transition state) are affected by the enhancement of helical propensities, thus, native-like local interactions appear to have a low specificity for the native conformation; (2) our results support the idea, proposed from thermodynamic analysis of the mutants, that the denatured state under native conditions becomes more compact upon enhancement of helical propensities; (3) the rate of folding in aqueous solution decreases in all the mutants, suggesting that the optimization of the folding rate in this protein requires low secondary structure propensities. Regarding the description of the folding pathway of Che Y, we find evidence that the folding transition state of Che Y is constituted by two sub-domains with different degree of helical structure. The first includes helices 1 and 2 which are rather structured, while the second encompasses the last three helices, which are very unstructured. On the other hand, the same analysis for the folding intermediate indicates that all the five alpha-helices are, on average, rather structured. Thus, suggesting that a large structural reorganization of the last three alpha-helices must take place before folding can be completed. This conclusion indicates that the folding intermediate of Che Y is a misfolded species.
Subject(s)
Bacterial Proteins/chemistry , Membrane Proteins/chemistry , Protein Folding , Bacterial Proteins/genetics , Escherichia coli , Kinetics , Membrane Proteins/genetics , Methyl-Accepting Chemotaxis Proteins , Mutation , ThermodynamicsABSTRACT
BACKGROUND: Protein engineering analysis has been used as a tool to determine the structure of the transition state of two different proteins: CI-2 and barnase. CI-2 belongs to the group of small, globular proteins with no disulphide bonds that fold via a two-state mechanism. Barnase is a larger protein (110 aa) and displays a folding intermediate. The structure of the transition state of both proteins is quite different. Whereas in CI-2 no region is fully native and it looks like an expanded form of the folded state, in barnase several regions are folded and the rate-limiting step seems to be the consolidation of the hydrophobic core. On the basis of these results, a unified scheme for the transition state of protein folding has been presented. We decided to characterize the folding pathway, or pathways, present in the alpha/beta parallel family of proteins using one of the smallest members, CheY (129 aa), as a model case. RESULTS: The folding pathway of CheY contains, as does that of barnase, a kinetic intermediate. The picture obtained for CheY from the equilibrium and kinetic analyses of several mutations scattered throughout the whole protein is different from that found for barnase. On the basis of the experimental results and the structure of CheY, the protein can be divided into two subdomains (from beta-strand 1 to beta-strand 3 and from beta-strand 3 to the C terminus). Whereas the structure of the first subdomain in the transition state resembles that found for the CI-2 protein, the second subdomain is compact but unstructured. The packing of the first alpha-helix against beta-strands 1 and 2 seems to be the nucleus around which the rest of the protein folds. CONCLUSIONS: Comparison of the transition state of barnase with those of CheY and CI-2 indicates that different proteins have different transition states, probably depending on the energetics and the position of the rate-limiting step in the folding pathway. CheY appears to fold through a nucleation/condensation mechanism as has been found for CI-2. The rate-determining step in some multimodular proteins could be the formation of a stable domain, with the less stable domains folding after the major rate-determining step.
Subject(s)
Bacterial Proteins , Membrane Proteins/chemistry , Peptides/chemistry , Amino Acid Sequence , Chymotrypsin/antagonists & inhibitors , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins , Kinetics , Membrane Proteins/genetics , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Peptides/genetics , Plant Proteins , Protein Engineering , Protein Folding , Protein Structure, Secondary , Ribonucleases/chemistry , Ribonucleases/genetics , ThermodynamicsABSTRACT
BACKGROUND: Protein stability appears to be governed by non-covalent interactions. These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions). The specific role of local interactions is controversial. Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins. However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability. Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability. RESULT: To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity. alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing. The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides. Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ([urea]1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m). ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f). CONCLUSIONS: These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state. Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects. Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions.
Subject(s)
Bacterial Proteins/chemistry , Membrane Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Stability , Escherichia coli/chemistry , Escherichia coli/genetics , Kinetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Denaturation/drug effects , Protein Folding , Protein Structure, Secondary , Thermodynamics , Urea/pharmacologySubject(s)
Adrenal Gland Neoplasms/diagnosis , Adrenal Gland Neoplasms/complications , Adrenal Gland Neoplasms/etiology , Adrenal Gland Neoplasms/secondary , Adrenal Gland Neoplasms/surgery , Adrenal Glands/diagnostic imaging , Adrenal Glands/pathology , Biopsy, Needle , Cushing Syndrome/diagnosis , Cushing Syndrome/etiology , Cytodiagnosis , Humans , Magnetic Resonance Imaging , Radionuclide Imaging , Tomography, X-Ray ComputedABSTRACT
A series of Ala vs. Gly mutations at different helical and nonhelical positions of the chemotactic protein CheY, from E. coli, has been made. We have used this information to fit a general analytical equation that describes the free energy changes of an Ala to Gly mutation within +/- 0.45 kcal mol-1 with 95% confidence. The equation includes three terms: (1) the change in solvent-accessible hydrophobic surface area, corrected for the possible closure of the cavity left by deleting the C beta of the Ala; (2) the change in hydrophilic area of the nonintramolecularly hydrogen-bonded groups; and (3) the dihedral angles of the position being mutated. This last term extends the calculation to any conformation, not only alpha-helices. The general applicability of the equation for Ala vs. Gly mutations, when Ala or a small solvent-exposed polar residue is the wild-type residue, has been tested using data from other proteins: barnase, CI2 trypsin inhibitor, T4 lysozyme, and Staphylococcus nuclease. The predictive power of this simple approach offers the possibility of extending it to more complex mutations.
Subject(s)
Alanine/chemistry , Bacterial Proteins/chemistry , Glycine/chemistry , Membrane Proteins/chemistry , Protein Structure, Secondary , Bacterial Proteins/genetics , Computer Simulation , Escherichia coli/chemistry , Escherichia coli Proteins , Hydrogen Bonding , Membrane Proteins/genetics , Methyl-Accepting Chemotaxis Proteins , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Recombinant Proteins/chemistry , ThermodynamicsABSTRACT
Backgound. Protein engineering analysis has been used as a tool to determine the structure of the transition state of two different proteins: CI-2 and barnase. CI-2 belongs to the group of small, globular proteins with no disulphide bonds that fold via a two-state mechanism. Barnase is a larger protein (110 aa) and displays a folding intermediate. The structure of the transition state of both proteins is quite different. Whereas in CI-2 no region is fully native and it looks like an expanded form of the folded state, in barnase several regions are folded and the rate-limiting step seems to be the consolidation of the hydrophobic core. On the basis of these results, a unified scheme for the transition state of protein folding has been presented. We decided to characterize the folding pathway, or pathways, present in the alpha/beta parallel family of proteins using one of the smallest members, CheY (129 aa), as a model case. Results. The folding pathway of CheY contains, as does that of barnase, a kinetic intermediate. The picture obtained for CheY from the equilibrium and kinetic analyses of several mutations scattered throughout the whole protein is different from that found for barnase. On the basis of the experimental results and the structure of CheY, the protein can be divided into two subdomains (from beta-strand 1 to beta-strand 3 and from beta-strand 3 to the C terminus). Whereas the structure of the first subdomain in the transition state resembles that found for the CI-2 protein, the second subdomain is compact but unstructured. The packing of the first alpha-helix against beta-strands 1 and 2 seems to be the nucleus around which the rest of the protein folds. Conclusion. Comparison of the transition state of barnase with those of CheY and CI-2 indicates that different proteins have different transition states, probably depending on the energetics and the position of the rate-limiting step in the folding pathway. CheY appears to fold through a nucleation/condensation mechanism as has been found for CI-2. The rate-determining step in some multi-modular proteins could be the formation of a stable domain, with the less stable domains folding after the major rate-determining step.
