The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

Phosphatidylinositol 3‐phosphate (PtdIns3P) orchestrates endosomal cargo transport, fusion and motility by recruiting FYVE or PX domain‐containing effector proteins to endosomal membranes. In an attempt to discover novel PtdIns3P effectors involved in the termination of growth factor receptor signalling, we performed an siRNA screen for epidermal growth factor (EGF) degradation, targeting FYVE and PX domain proteins in the human proteome. This screen identified several potential regulators of EGF degradation, including HRS (used as positive control), PX kinase, MTMR4 and Phafin2/PLEKHF2. As Phafin2 has not previously been shown to be required for EGF receptor (EGFR) degradation, we performed further functional studies on this protein. Loss of Phafin2 was found to decrease early endosome size, whereas overexpression of Phafin2 resulted in enlarged endosomes. Moreover, both the EGFR and the fluid‐phase marker dextran were retained in abnormally small endosomes in Phafin2‐depleted cells. In yeast two‐hybrid analysis we identified Phafin2 as a novel interactor of the endosomal‐tethering protein EEA1, and Phafin2 colocalized strongly with EEA1 in microdomains of the endosome membrane. Our results suggest that Phafin2 controls receptor trafficking and fluid‐phase transport through early endosomes by facilitating endosome fusion in concert with EEA1.     

Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.     

Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.     

Hydrogen peroxide affects abscisic acid binding to ABAP1 in barley aleurones.

Dramatic increases in H2O2 levels have been observed following abscisic acid (ABA) treatment of plant tissues. Following ABA treatment in aleurone cells, H2O2 reached transient levels of approximately 115 micromol/L H2O2. To determine whether ABA perception was modified by such changes, the effect of H2O2 on a recently characterized ABA-binding protein (ABAP1), cloned from barley aleurone layers, was examined. ABA binding to the protein was weakened by H2O2 in a concentration-dependent manner. A concentration of 75 micromol/L H2O2 gave a 50% decline in ABA binding in a reaction following first-order kinetics, indicative of binding-site susceptibility to its microenvironment. We monitored the unfolding of ABAP1 using steady-state and time-resolved tryptophan fluorescence, while following the capacity of ABAP1 to bind ABA. ABA binding decreased by 50% following ABAP1 denaturation with 1 mol/L guanidine hydrochloride or 2 mol/L urea, while the maximum emission spectra (lambda emi) red shifted from 338 to 347 nm at 3.5 mol/L guanidine hydrochloride and 5 mol/L urea. However, only a slight blue shift of lambda emi was observed following either ABAP1 incubation with H2O2 or binding to (+)-ABA (physiologically active ABA). The equilibrium ABA dissociation rate accelerated in the presence of 250 micromol/L H2O2, with the half-time dissociation reduced to 8 min. A comparison of inactivation kinetics and conformational changes shows that inactivation of ABAP1 occurs before any noticeable conformational change. This suggests that the ABA binding site is highly responsive to its microenvironment and is situated in a region that is more flexible than the protein molecule as a whole. The results demonstrate that H2O2, generated by ABA treatment of aleurone layers, is sufficient to affect the ABA-binding capacity of ABAP1, suggesting that this may be another level of control of ABA signal transduction.     

Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has α-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.     

Protein stability: functional dependence of denaturational Gibbs energy on urea concentration

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.     

Denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions

The denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions was examined by monitoring changes in the intrinsic fluorescence of tryptophan and tyrosyl residues. Changes in various fluorescence parameters, such as quantum yield, emission maximum, spectral half-width, fluorescence depolarization, and fluorescence quenching by acrylamide, have indicated that while phaseolin is relatively stable up to 8 M urea, it is completely destabilized in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate. Furthermore, while the denaturation of phaseolin in urea solutions followed a two-step process, that in guanidine hydrochloride and sodium dodecyl sulfate followed a single-step process. While the accessibility of tryptophan residues to the nonionic acrylamide quencher is almost 100% in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate, only about 72% was accessible in 8 M urea compared to 52% in native phaseolin. The results presented here suggest that the protomeric structure of phaseolin is quite stable to changes in the environment. This structural stability may be partly responsible for its resistance to proteolysis by various proteinases.     

A guanidine hydrochloride induced change in ribonuclease without gross unfolding

Although denaturation of ribonuclease by guanidine hydrochloride to a random coil has been considered to be a simple two-state mechanism, the time dependence of our calorimetric data indicate that a cooperative endothermic pretransition may occur near 1.25 M. guanidine hydrochloride (pH 6 and 25°C) without gross unfolding of the protein. Reexamination of other observables as a function of guanidine hydrochloride concentrations as well as activity measurements suggests the possibility of some process other than simple binding occurring in the concentration range below the onset of gross denaturation.     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

Physicochemical properties of thiol proteinase inhibitor isolated from goat pancreas

Thiol proteinase inhibitors are crucial to proper functioning of all living tissues consequent to their cathepsin regulatory and myriad important biologic properties. Equilibrium denaturation of dimeric goat pancreas thiol proteinase inhibitor (PTPI), a cystatin superfamily variant has been studied by monitoring changes in the protein's spectroscopic and functional characteristics. Denaturation of PTPI in guanidine hydrochloride and urea resulted in altered intrinsic fluorescence emission spectrum, diminished negative circular dichroism, and loss of its papain inhibitory potential. Native like spectroscopic properties and inhibitory activity are only partially restored when denaturant is diluted from guanidine hydrochloride unfolded samples demonstrating that process is partially reversible. Coincidence of transition curves and dependence of transition midpoint (3.2M) on protein concentration in guanidine hydrochloride‐induced denaturation are consistent with a two‐state model involving a native like dimer and denatured monomer. On the contrary, urea‐induced unfolding of PTPI is a multiphasic process with indiscernible intermediates. The studies demonstrate that functional conformation and stability are governed by both ionic and hydrophobic interactions. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 708–717, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation

The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1-10. Equilibrium unfolding with urea showed that values of D(1/2) (5.7 M) and DeltaG(H(2)O) (12.8 kcal/mol) were highest at pH 5.0, its pI and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of DeltaG(H(2)O) although GdnHCl was 2-fold more potent denaturant with D(1/2) value of 3.1 M. The curves of fraction unfolded (f(U)) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0-3.0 and 6.0-9.0. Thermal denaturation in the pH range 4.0-5.5 resulted in aggregation and precipitation of the protein above 55 degrees C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission lambda(max) and enhancement of fluorescence intensity of ANS at 70 degrees C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with alpha-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge-charge repulsion at the pI of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F right arrow over left arrow U mechanism. The pH dependence of stability described by DeltaG(H(2)O) (urea) and DeltaG (25 degrees C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds.     

Bromophenol blue binding as a probe to study urea and guanidine hydrochloride denaturation of bovine serum albumin

Urea and guanidine hydrochloride (GdnHCl) denaturation of bovine serum albumin (BSA) were investigated using bromophenol blue (BPB) binding as a probe. Addition of BPB to BSA produced an absorption difference spectrum in the wavelength range, 525-675 nm with a minimum at 587 nm and a maximum at 619 nm. The magnitude of absorption difference (DeltaAbs.) at 619 nm decreased on increasing urea/GdnHCl concentration and followed the denaturation curve. The denaturation was found to be a two-state, single-step transition. The transitions started at 1.75 and 0.875 M and completed at 6.5 and 3.25 M with the mid point occurring around 4.0 and 1.5 M urea and GdnHCl concentrations, respectively. The value of free energy of stabilization, DeltaGDH2O as determined from urea and GdnHCl denaturation curves was found to be 4041 and 4602 cal/mol, respectively. Taken together, these results suggest that BPB binding can be used as a probe to study urea and GdnHCl denaturation of BSA.     

Axial ligand effects on myoglobin stability

Reversible guanidine hydrochloride denaturation has been applied to obtain the first quantitative estimate of ligand-induced changes in hemoprotein conformational free energy. It is found that strong field (low spin) complexes, e.g. cyanometmyoglobin (MbCN) and azido metmyoglobin (MbN3), are 1.0 +/- 0.1 kcal/mol more stable than the high spin analogs aquometmyoglobin (MbH2O) and fluorometmyoglobin (MbF). This observed stability increment is essentially independent of the model chosen for data analysis. These results demonstrate the value of denaturation titration in measuring the stabilization of hemoprotein conformation by ligand binding. The denaturation of MbN3 appears complex. This complexity may be quantitatively accounted for by considering spin state equilibria. Applying this correction, MbCN and MbN3 have essentially the same stability in spite of steric differences in the two proteins. This result implies metal spin state is more important than ligand stereochemistry in determining the conformational free energy of myoglobin.     

The phox homology (PX) domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal compartment is required for its ability to regulate epidermal growth factor receptor degradation

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (PtdIns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6767-6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P(3) in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) but not to PtdIns(3,4,5)P(3). To address the significance of PtdIns(3,4,5)P(3) binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P(3) levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P(2) being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we have shown that the PX domain-dependent/early endosomal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.     

Comparison of denaturation of tryptophan synthase alpha-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme.     

Denaturation induced aggregation in α‐crystallin: differential action of chaotropes

α‐Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α‐crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α‐crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α‐crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α‐crystallin at 216 nm, suggesting gain in β‐sheet structure in the presence of GdnHCl (0.5–1 M) followed by unfolding at higher concentration (2–6 M). However, only minor changes in the secondary structure of α‐crystallin were observed in the presence of urea. Moreover, 8‐anilinonaphthalene‐1‐sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α‐crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α‐crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α‐crystallin in the presence of GdnHCl, whereas only aggregate‐like structures were observed in α‐crystallin treated with urea. Our results suggest that α‐crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.     

Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.     

The role of the intrachain disulfide bond in the conformation and stability of the constant fragment of the immunoglobulin light chain.

The conformation and stabilities of the CL fragment isolated from a type lambda Bence Jones protein and the fragment in which the intrachain disulfide bond had been reduced were studied by measuring CD, fluorescence, and ultraviolet absorption. The results indicated that no great conformational change occurs on reduction of the disulfide, unless the SH groups are alkylated. Intact CL was more resistant than reduced CL to guanidine hydrochloride. The denaturation curves were analyzed using an equation based on the binding of guanidine hydrochloride and the free energy changes of denaturation in the absence of the denaturant were estimated as about 6 kcal.mol-1 for intact CL and about 1.8 kcal.mol-1 for reduced CL. The difference in stability between intact CL and reduced CL was explained to a great extent in terms of the entropy change associated with reduction of the intrachain disulfide bond of the fragment in the denatured state.     

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.