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Alain Doucet

Publications and source records attributed to Alain Doucet.

At least 19 recordsLinked to original sources
PubMed2006

Kidney collecting duct acid-base "regulon".

Kidneys are essential for acid-base homeostasis, especially when organisms cope with changes in acid or base dietary intake. Because collecting ducts constitute the final site for regulating urine acid-base balance, we undertook to identify the gene network involved in acid-base transport and regulation in the mouse outer medullary collecting duct (OMCD). For this purpose, we combined kidney functional studies and quantitative analysis of gene expression in OMCDs, by transcriptome and candidate gene approaches, during metabolic acidosis. Furthermore, to better delineate the set of genes concerned with acid-base disturbance, the OMCD transcriptome of acidotic mice was compared with that of both normal mice and mice undergoing an adaptative response through potassium depletion. Metabolic acidosis, achieved through an NH4Cl-supplemented diet for 3 days, not only induced acid secretion but also stimulated the aldosterone and vasopressin systems and triggered cell proliferation. Accordingly, metabolic acidosis increased the expression of genes involved in acid-base transport, sodium transport, water transport, and cell proliferation. In particular, >25 transcripts encoding proteins involved in urine acidification (subunits of H-ATPase, kidney anion exchanger, chloride channel Clcka, carbonic anhydrase-2, aldolase) were co-regulated during acidosis. These transcripts, which cooperate to achieve a similar function and are co-regulated during acidosis, constitute a functional unit that we propose to call a "regulon".

Acid-Base Equilibrium↗
PubMed2006

Increased local levels of granulocyte colony-stimulating factor are associated with the beneficial effect of pre-elafin (SKALP/trappin-2/WAP3) in experimental emphysema.

Few therapeutic options are offered to treat inflammation and alveolar wall destruction in emphysema. The effect of recombinant human pre-elafin, an elastase inhibitor, was evaluated in porcine pancreatic elastase (PPE)-induced emphysema in C57BL/6 mice. In a first protocol, mice received a single instillation of pre-elafin (17.5 pmol/mouse) at 1 h post-PPE and were sacrificed up to 72 h post-PPE. A single instillation of pre-elafin significantly reduced PPE-induced neutrophil accumulation in lungs, as assessed by bronchoalveolar lavage (BAL), by 51%, 71% and 67% at 24, 48 and 72 h, respectively. In a second protocol, mice also received a single dose of PPE, but pre-elafin three times a week for 2 weeks. After 2 weeks, pre-elafin significantly reduced the PPE-induced increase in BAL macrophage numbers, airspace dimensions and lung hysteresivity by 74%, 62% and 52%, respectively. Since G-CSF was previously shown to reduce emphysematous changes in mice, the BAL levels of this mediator were measured 6 h post-PPE in animals treated as described in the first protocol. Pre-elafin significantly increased G-CSF levels in PPE-exposed mice compared to sham- and PPE only-exposed animals. This suggests that the beneficial effects of pre-elafin could be mediated, at least in part, by its ability to increase G-CSF levels in the lung.

Animals↗
PubMed2005

Hyperaldosteronemia and activation of the epithelial sodium channel are not required for sodium retention in puromycin-induced nephrosis.

Edema and ascites in nephrotic syndrome mainly result from increased Na+ reabsorption along connecting tubules and cortical collecting ducts (CCD). In puromycin aminonucleoside (PAN)-induced nephrosis, increased Na+ reabsorption is associated with increased activity of the epithelial sodium channel (ENaC) and Na+,K+-ATPase, two targets of aldosterone. Because plasma aldosterone increases in PAN-nephrotic rats, the aldosterone dependence of ENaC activation in PAN nephrosis was investigated. For this purpose, (1) the mechanism of ENaC activation was compared in nephrotic and sodium-depleted rats, and (2) ENaC activity in PAN-nephrotic rats was evaluated in the absence of hyperaldosteronemia. The mechanism of ENaC activation was similar in CCD from nephrotic and sodium-depleted rats, as demonstrated by (1) increased number of active ENaC evaluated by patch clamp, (2) recruitment of ENaC to the apical membrane determined by immunohistochemistry, (3) shift in the electrophoretic profile of gamma-ENaC, and (4) increased abundance of beta-ENaC mRNA. Corticosteroid clamp fully prevented all PAN-induced changes in ENaC but did not alter the development of a full-blown nephrotic syndrome with massive albuminuria, amiloride-sensitive sodium retention, induction of CCD Na+,K+-ATPase, and ascites. It is concluded that in PAN-nephrosis, (1) ENaC activation in CCD is secondary to hyperaldosteronemia, (2) sodium retention and induction of Na+,K+-ATPase in CCD are independent of hyperaldosteronemia, and (3) ENaC is not necessarily limiting for sodium reabsorption in the distal nephron.

Animals↗
PubMed2005

Molecular identification of Sch28080-sensitive K-ATPase activities in the mouse kidney.

Rat collecting ducts display either an ouabain-insensitive or an ouabain-sensitive K-ATPase activity inhibited by Sch28080 according as animals are fed a normal or a potassium-depleted diet (types I and III K-ATPase, respectively). Two isoforms of H,K-ATPase have been cloned from rat gastric mucosa and colon, respectively. Gastric and colonic H,K-ATPase are expressed in the kidney, suggesting that they might account for types I and III K-ATPases. However, this hypothesis is not fully supported by segmental expression of gastric and colonic H,K-ATPase along the rat collecting duct, as well as by comparison of the pharmacological properties of gastric and colonic H,K-ATPase expressed in Xenopus ovocyte and types I and III K-ATPases in rat collecting ducts. The aim of the present work is to address directly the molecular origin of types I and III K-ATPases in the mouse collecting duct by measuring K-ATPase activities in collecting ducts of wild-type mice and mice genetically deficient in either gastric or colonic H,K-ATPase fed either a regular or a potassium-depleted diet. Like the rat, mouse collecting ducts display type I or III K-ATPase activity when fed a regular or a potassium-depleted diet, respectively. Type I K-ATPase activity is detected in colonic H,K-ATPase-deficient mice but not in gastric H,K-ATPase-deficient animals. Conversely, type III K-ATPase activity disappears in colonic H,K-ATPase-deficient but not in gastric H,K-ATPase-deficient mice. In conclusion, types I and III K-ATPases measured in collecting ducts of normal and potassium-depleted mice reflect the functional expression of gastric and colonic H,K-ATPase, respectively.

Animals↗
PubMed2005

T-cell transcriptome analysis points up a thymic disorder in idiopathic nephrotic syndrome.

BACKGROUND: Idiopathic nephrotic syndrome is a proteinuric disease secondary to the release of a nonidentified circulating glomerular permeability factor by T cells. Because specificities of T-cell activation in idiopathic nephrotic syndrome remain unknown, we evaluated transcriptional activation of T cells in nephrotic patients during proteinuria. METHODS: Transcriptomes of CD2+ cells were analyzed by serial analysis of gene expression (SAGE) in a nephrotic child during proteinuria relapse and after remission, away from any immunosuppressive treatment. Expression of specific transcripts overexpressed during proteinuria relapse was compared by reverse transcription-polymerase chain reaction (RT-PCR) in CD2+ cells from 11 nephrotic patients during relapse and remission and 11 non nephrotic patients during infection and after recovery. RESULTS: Differential analysis of CD2+ cell transcriptome identified >200 mRNA tags overexpressed during proteinuria relapse, including many T-cell markers. RT-PCR analysis of expression of specific transcripts indicated that (1) under remission conditions, nephrotic children displayed induction of four transcripts, including IKBKB, and repression of NFKBIA as compared to non nephrotic children after recovery, and (2) proteinuria relapse was associated with induction of L-selectin and T-lymphocyte maturation-associated protein, two markers of T-cell differentiation and recent emigrant/naive T cells. CONCLUSION: Results indicate that circulating T cells from relapsing nephrotic patients include a significant population of low-mature cells while those from nephrotic patients in remission are characterized by constitutive activation of nuclear factor-kappaB (NF-kappaB), altogether suggesting a thymic dysregulation of apoptosis in nephrotic patients.

CD2 Antigens↗
PubMed2005

Expression of concentrative nucleoside transporters SLC28 (CNT1, CNT2, and CNT3) along the rat nephron: effect of diabetes.

BACKGROUND: The renal reabsorption of natural nucleosides and a variety of nucleoside-derived drugs relies on the function of the apically located, Na(+)-dependent, concentrative nucleoside transporters CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3). The aims of this study were to determine the segmental localization of the three SLC28 family members and to establish whether streptozotocin-induced diabetes alters their expression. METHODS: SLC28 expression was measured by real-time polymerase chain reaction (PCR) on microdissected sections of rat nephrons. Diabetes was induced by streptozotocin treatment and the biochemical profiles of control, diabetic, and insulin-treated rats were established. The effect of diabetes on SLC28 expression was assessed in those segments that significantly express SLC28 genes. RESULTS: CNT1-3 mRNAs were expressed in the proximal tubule and glomerulus. In addition, CNT2 and CNT3 mRNAs were expressed in the outer medullary and cortical collecting duct, respectively. Diabetes reduced expression of the three CNTs in almost all nephron segments, and this effect was not prevented by an insulin treatment that normalized all blood and urine parameters. Diabetes increased CNT1 and CNT3 expression in the glomerulus and insulin treatment decreased it. CONCLUSION: The relative distribution of SLC28 gene expression suggests a role for the proximal tubule in renal nucleoside clearance and an accessory role for CNT2 and CNT3, in adenosine-mediated regulation of collecting duct functions. Diabetes probably may impair nucleoside clearance independently of insulin.

Animals↗
PubMed2004

Global analysis of gene expression in mammalian kidney.

The past decade has seen the achievement of sequencing of human, rat and mouse genomes and the development of high-throughput methods for quantitative monitoring of gene expression. The aim of the beginning post-genomic era is to determine the function of all these genes, a challenge in which the community of physiologists should have a leading role. In this short review, we discuss the relevance, feasibility and impact of transcriptome analysis in renal physiology. Comparison of transcriptomes demonstrates that cell lines are poor physiological models, making it necessary to work on native kidney tissue. Palliating kidney heterogeneity therefore requires the development of methods for transcriptome analysis sensitive enough to be compatible with microdissected nephron segments. Axial comparison of transcriptomes along the human nephron unexpectedly points out that the segmentation of nephrons concerns not only genes involved in solute and water transport functions and their regulation, but also genes related to the control of cell division, differentiation and apoptosis. Comparison of transcriptome of a same nephron segment from mice under different pathophysiological conditions outlines the wide pleiotropy of kidney function regulations. Both types of comparative studies also identify yet unknown transcripts specifically expressed along the nephron or under pathophysiological conditions.

Animals↗
PubMed2004

ERK1/2 controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell of the cortical collecting duct of the mouse kidney.

The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 pathway as shown in vivo by immunostaining of phosphorylated active ERK1/2 (pERK1/2). The MEK1/2-ERK1/2 pathway controls many different ion transports both in proximal and distal nephron, raising the question of whether this pathway is involved in the basal and/or hormone-dependent transepithelial sodium reabsorption in the principal cell of the cortical collecting duct (CCD), a process mediated by the apical epithelial sodium channel and the basolateral sodium pump (Na,K-ATPase). To answer this question we used ex vivo microdissected CCDs from normal mouse kidney or in vitro cultured mpkCCDcl4 principal cells. Significant basal levels of pERK1/2 were observed ex vivo and in vitro. Aldosterone and vasopressin, known to up-regulate sodium reabsorption in CCDs, did not change ERK1/2 activity either ex vivo or in vitro. Basal and aldosterone- or vasopressin-stimulated sodium transport was down-regulated by the MEK1/2 inhibitor PD98059, in parallel with a decrease in pERK1/2 in vitro. The activity of Na,K-ATPase but not that of epithelial sodium channel was inhibited by MEK1/2 inhibitors in both unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface biotinylation showed that intrinsic activity rather than cell surface expression of Na,K-ATPase was controlled by pERK1/2. PD98059 also significantly inhibited the activity of Na,K-ATPase ex vivo. Our data demonstrate that the ERK1/2 pathway controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell and indicate that basal constitutive activity of the ERK1/2 pathway is a critical component of this control.

Aldosterone↗
PubMed2004

Plasticity of mouse renal collecting duct in response to potassium depletion.

Plasticity of mouse renal collecting duct in response to potassium depletion.--Renal collecting ducts are the main sites for regulation of whole body potassium balance. Changes in dietary intake of potassium induce pleiotropic adaptations of collecting duct cells, which include alterations of ion and water transport properties along with an hypertrophic response. To study the pleiotropic adaptation of the outer medullary collecting duct (OMCD) to dietary potassium depletion, we combined functional studies of renal function (ion, water, and acid/base handling), analysis of OMCD hypertrophy (electron microscopy) and hyperplasia (PCNA labeling), and large scale analysis of gene expression (transcriptome analysis). The transcriptome of OMCD was compared in mice fed either a normal or a potassium-depleted diet for 3 days using serial analysis of gene expression (SAGE) adapted for downsized extracts. SAGE is based on the generation of transcript-specific tag libraries. Approximately 20,000 tags corresponding to 10,000 different molecular species were sequenced in each library. Among the 186 tags differentially expressed (P < 0.05) between the two libraries, 120 were overexpressed and 66 were downregulated. The SAGE expression profile obtained in the control library was representative of different functional classes of proteins and of the two cell types (principal and alpha-intercalated cells) constituting the OMCD. Combined with gene expression analysis, results of functional and morphological studies allowed us to identify candidate genes for distinct physiological processes modified by potassium depletion: sodium, potassium, and water handling, hyperplasia and hypertrophy. Finally, comparison of mouse and human OMCD transcriptomes allowed us to address the question of the relevance of the mouse as a model for human physiology and pathophysiology.

Acid-Base Equilibrium↗
PubMed2004

Differential regulation of collecting duct Na+, K+-ATPase and K+ excretion by furosemide and piretanide: role of bradykinin.

In response to chronic treatment with furosemide, collecting ducts adapt their function to the initial loss of Na+ to prevent further Na+ loss and extracellular volume decrease. This adaptation, which includes the overexpression of Na+, K+-ATPase, is thought to account for most of the kaliuretic effect of furosemide. Because piretanide is reported to be less kaliuretic than equidiuretic doses of furosemide, the authors compared the effects of 1-wk treatment with the two loop diuretics on urinary potassium excretion and on Na+, K+-ATPase activity in the collecting duct. At equidiuretic and equinatriuretic doses, furosemide increased urinary potassium excretion as well as collecting duct Na+, K+-ATPase activity, whereas piretanide had no effect on either parameter. These effects of furosemide were curtailed by concomitant administration of the angiotensin-converting enzyme inhibitor enalapril, but they were not altered either by clamping changes in plasma aldosterone or by blocking type I angiotensin receptors. Treatment with the antagonist of bradykinin B2 receptors Hoe140 mimicked the two effects of furosemide. In addition, the effects of Hoe140 and furosemide were not additive. Finally, piretanide increased urinary bradykinin excretion, whereas furosemide did not. These results suggest that induction of collecting duct Na+, K+-ATPase (a) accounts for the kaliuretic effect of furosemide, (b) is independent of the renin/angiotensin/aldosterone system, (c) results from increased Na+ delivery to the collecting duct and enhanced intracellular Na+ concentration, and (d) is prevented in piretanide treated rats by increased bradykinin production that may limit apical Na+ entry in collecting duct principal cells.

Animals↗
PubMed2003

A panoramic view of gene expression in the human kidney.

To gain a molecular understanding of kidney functions, we established a high-resolution map of gene expression patterns in the human kidney. The glomerulus and seven different nephron segments were isolated by microdissection from fresh tissue specimens, and their transcriptome was characterized by using the serial analysis of gene expression (SAGE) method. More than 400,000 mRNA SAGE tags were sequenced, making it possible to detect in each structure transcripts present at 18 copies per cell with a 95% confidence level. Expression of genes responsible for nephron transport and permeability properties was evidenced through transcripts for 119 solute carriers, 84 channels, 43 ion-transport ATPases, and 12 claudins. Searching for differences between the transcriptomes, we found 998 transcripts greatly varying in abundance from one nephron portion to another. Clustering analysis of these transcripts evidenced different extents of similarity between the nephron portions. Approximately 75% of the differentially distributed transcripts corresponded to cDNAs of known or unknown function that are accurately mapped in the human genome. This systematic large-scale analysis of individual structures of a complex human tissue reveals sets of genes underlying the function of well-defined nephron portions. It also provides quantitative expression data for a variety of genes mutated in hereditary diseases and helps in sorting candidate genes for renal diseases that affect specific portions of the human nephron.

Cluster Analysis↗
PubMed2003

Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arterioles of rat kidney.

ANG II controls the vascular tone of pre- and postglomerular arterioles, and thereby glomerular filtration, through binding to either AT1A, AT1B, or AT2 receptors. AT1 receptors, which are coupled to intracellular Ca2+ signaling, have vasoconstricting effects, whereas AT2 receptors, whose signaling mechanism is unknown, induce vasodilatation. The angiotensin receptors have been characterized in afferent arterioles, which express the three types of receptors, but not in efferent arterioles. Two subpopulations of juxtamedullary efferent arterioles, muscular ones which terminate as vasa rectae and thin ones which terminate as peritubular capillaries, have been described. They display functional heterogeneity with regard to the ANG II response. To evaluate whether these differences are associated with differential expression of ANG II receptors, we examined the expression pattern of AT1A, AT1B, and AT2 receptor mRNAs by RT-PCR in these arterioles and studied the effect of valsartan, a specific AT1-receptor antagonist. Results indicate that muscular arterioles express AT1A, AT1B, and AT2 receptors, whereas thin arterioles only express the AT1A and AT2 types, and at a much lower level. Valsartan fully inhibited ANG II-induced increases in intracellular Ca2+ in both arteriolar types, but with different kinetics. In muscular arterioles, inhibition was monoexponential, whereas it displayed a marked positive cooperativity in thin arterioles. Finally, the apparent affinity for valsartan was higher in muscular than in thin arterioles. In conclusion, this study further documents the differences between muscular and thin efferent arterioles with regard to ANG II signalization in the rat kidney.

Angiotensin II↗
PubMed2003

Intracellular Na+ controls cell surface expression of Na,K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells.

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K(+)-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.

Amphotericin B↗
PubMed2003

Mechanism of control of Na,K-ATPase in principal cells of the mammalian collecting duct.

The collecting duct is the site of final Na reabsorption according to Na balance requirements. Using isolated rat cortical collecting ducts (CCD) and mpkCCD(cl4) cells, a mouse cortical collecting duct cell line, we have studied the physiological control of Na,K-ATPase, the key enzyme that energizes Na reabsorption. Aldosterone, a major regulator of Na transport by the collecting duct, stimulates Na,K-ATPase activity through both recruitment of intracellular pumps and increased total amounts of Na pump subunits. This effect is observed after a lag time of 1 hour and is independent of Na entry through ENaC, but requires de novo transcription and translation. Vasopressin and cAMP, its second messenger, stimulate Na,K-ATPase activity within minutes through translocation of Na pumps from a brefeldin A-sensitive intracellular pool to the plasma membrane. Dysregulation of collecting duct Na,K-ATPase activity is at least in part responsible of the Na retention observed in nephritic syndrome. In this setting, Na,K-ATPase activity and subunit synthesis are specifically increased in CCD. In conclusion, aldosterone, vasopressin, and intracellular Na control the cell surface expression of Na,K-ATPase and translocation from intracellular stores is a major mechanism of regulation of Na,K-ATPase activity in collecting duct principal cells.

Aldosterone↗
PubMed2003

Mechanism of activation of ERK and H-K-ATPase by isoproterenol in rat cortical collecting duct.

Isoproterenol stimulates H-K-ATPase activity in rat cortical collecting duct beta-intercalated cells through a PKA-dependent pathway. This study aimed at determining the signaling pathway underlying this effect. H-K-ATPase activity was determined in microdissected collecting ducts preincubated with or without specific inhibitors or antibodies against intracellular signaling proteins. Transient cell membrane permeabilization with streptolysin-O allowed intracellular access to antibodies. Isoproterenol increased phosphorylation of ERK in a PKA-dependent manner, and inhibition of the ERK phosphorylation prevented the stimulation of H-K-ATPase. Antibodies against the monomeric G protein Ras or the kinase Raf-1 curtailed the stimulation of H-K-ATPase by isoproterenol, whereas antibodies against the related proteins Rap-1 and B-Raf had no effect. Pertussis toxin and inhibition of tyrosine kinases with genistein also curtailed isoproterenol-induced stimulation of H-K-ATPase. It is proposed that activation of PKA by isoproterenol induces the phosphorylation of beta-adrenergic receptors and the switch from G(s) to G(i) coupling. In turn, betagamma-subunits released from G(i) would activate a tyrosine kinase-Ras-Raf-1 pathway, leading to the activation of ERK1/2 and of H-K-ATPase.

Adrenergic beta-Agonists↗
PubMed2002

Protein kinase A-independent activation of ERK and H,K-ATPase by cAMP in native kidney cells: role of Epac I.

This study aimed at determining the signaling pathways underlying calcitonin- and isoproterenol-induced stimulation of H,K-ATPase in rat renal collecting duct. H,K-ATPase activity was determined in microdissected collecting ducts preincubated with or without either specific inhibitors or antibodies directed against intracellular signaling proteins. Transient cell membrane permeabilization with streptolysin-O allowed intracellular access of antibodies. The stimulation of H,K-ATPase by calcitonin and isoproterenol was mimicked by cAMP analogues and was abolished by adenylyl cyclase inhibition. Protein kinase A inhibition abolished isoproterenol but not calcitonin effect on H,K-ATPase. Calcitonin increased the phosphorylation of extracellular signal-regulated kinase (ERK) in a protein kinase A-independent manner, and the inhibition of the ERK phosphorylation prevented the stimulation of H,K-ATPase by calcitonin. Antibodies directed against either the cAMP-activated guanine-nucleotide exchange factor Epac I, the monomeric G protein Rap-1 or the kinase Raf-B, curtailed the stimulation of H,K-ATPase by calcitonin, whereas antibodies against the related monomeric G protein Ras or kinase Raf-1 had no effect. In conclusion, calcitonin stimulates H,K-ATPase through a cAMP/Epac I/Rap-1/Raf-B/ERK cascade.

Animals↗
PubMed2002

Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.

Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.

Circular Dichroism↗