Characterisation of the effect of electrostatic interaction on the structure of Trp-cage using molecular dynamics simulation

A mutated variant of 20 amino acid miniprotein Trp-cage (TC5b), called TC5c (Asp9 replaced by Asn9), was designed to demonstrate the effect of a salt bridge. As a result of strong electrostatic interaction, the distance distribution between Asp9 and Arg16 exhibited a larger probability in the range of the salt bridge for TC5b compared to TC5c. The probability of α-helix formation for residues 3–8, as well as for residues 11–14, was high for TC5b. The salt bridge formation between Asp9 and Arg16 in TC5b was indicated by (a) a strong correlation of their distance of separation with the subtended angle with the centre and (b) a step decrease in the distance between Gly11O and Arg16H at 12 ns. Replica exchange molecular dynamics simulation at different temperatures in the range of 270–590 K indicated that the average distance between Asp9 and Arg16, end-to-end distance, root mean square deviation with respect to a reference NMR structure of TC5b did not change significantly with temperature below 370 K for TC5b and increased at higher temperatures. These values were higher for TC5c for the whole temperature range, with their rate of increase with temperature being higher below 370 K.     

Reversing the typical pH stability profile of the Trp-cage

The Trp-cage, an 18-20 residue miniprotein, has emerged as a primary test system for evaluating computational fold prediction and folding rate determination efforts. As it turns out, a number of stabilizing interactions in the Trp-cage folded state have a strong pH dependence; all prior Trp-cage mutants have been destabilized under carboxylate-protonating conditions. Notable among the pH dependent stabilizing interactions within the Trp-cage are: (1) an Asp as the helix N-cap, (2) an H-bonded Asp9/Arg16 salt bridge, (3) an interaction between the chain termini which are in close spatial proximity, and (4) additional side chain interactions with Asp9. In the present study, we have prepared Trp-cage species that are significantly more stable at pH 2.5 (rather than 7) and quantitated the contribution of each interaction listed above. The Trp-cage structure remains constant with the pH change. The study has also provided measures of the stabilizing contribution of indole ring shielding from surface exposure and the destabilizing effects of an ionized Asp at the C-terminus of an α-helix.     

Cooperation between a salt bridge and the hydrophobic core triggers fold stabilization in a Trp-cage miniprotein

Miniproteins are adequate models to study various protein-structure modifying effects such as temperature, pH, point mutation(s), H-bonds, salt bridges, molecular packing, etc. Tc5b, a 20-residue Trp-cage protein is one of the smallest of such models with a stable 3D fold (Neidigh J. W. et al. (2002) Nat. Struct. Biol. 9, 425-430). However, Tc5b exhibits considerable heat-sensitivity and is only stable at relatively low temperatures. Here we report a systematic investigation of structural factors influencing the stability of Tc5b by solving its solution structure in different environments, varying temperature, and pH. The key interactions identified are the hydrophobic stacking of the aromatic rings of Tyr3 and Trp6 and the salt bridge formed between Asp9 and Arg18. To verify the importance of these interactions, selected variants (mutated, glycosylated and truncated) of Tc5b were designed, prepared, and investigated by NMR. Indeed, elimination of either of the key interactions highly destabilizes the structure. These observations enabled us to design a new variant, Tc6b, differing only by a methylene group from Tc5b, in which both key interactions are optimized simultaneously. Tc6b exhibits enhanced heat stability and adopts a stable fold at physiological temperature.     

Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus

Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.     

Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS

A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [³H]DA and inhibitor [³H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [³H]DA and [³H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.     

Contribution of a salt bridge to the thermostability of adrenodoxin determined by site-directed mutagenesis.

We identified a unique conserved salt bridge Arg89-Glu74 inside the protein core of adrenodoxin, which ensures proper orientation between the [2Fe-2S] cluster-containing domain and the recognition helix. Incorporation and geometry of the redox center were essentially preserved in the mutants E74D, R89A, and R89K as judged by EPR spectroscopy. However, absorption and CD spectra pointed out essential conformational changes in the protein vicinity of the [2Fe-2S] cluster. Judged by essentially increased K(m) and K(d) values and changed redox properties, mutations resulted in displacement of the recognition helix and hindered proper docking of the protein with both adrenodoxin reductase and CYP11A1. Substitutions of Arg89 and Glu74 induce thermodynamic destabilization attested by dramatically decreased unfolding temperature (T(d)) and enthalpy (Delta(d)H(T(d))). The heat capacity change of denaturation (Delta(d)C(p)) was significantly decreased for the mutants, suggesting that parts of the polypeptide chain normally hidden inside the protein core are exposed to the solvent in these variants.     

Role of residues in the adenosine binding site of NAD of the Ascaris suum malic enzyme

Ascaris suum mitochondrial malic enzyme catalyzes the divalent metal ion dependent conversion of l-malate to pyruvate and CO(2), with concomitant reduction of NAD(P) to NAD(P)H. In this study, some of the residues that form the adenosine binding site of NAD were mutated to determine their role in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the K(m) for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/E(t) decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/E(t), contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.     

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.     

Significance of local electrostatic interactions in staphylococcal nuclease studied by site-directed mutagenesis

In this paper, we show that amino acids Glu(73) and Asp(77) of staphylococcal nuclease cooperate unequally with Glu(75) to stabilize its structure located between the C-terminal helix and beta-barrel of the protein. Amino acid substitutions E73G and D77G cause losses of the catalytic efficiency of 24 and 16% and cause thermal stability losses of 22 and 26%, respectively, in comparison with the wild type (WT) protein. However, these changes do not significantly change global and local secondary structures, based on measurements of fluorescence and CD(222 nm). Furthermore, x-ray diffraction analysis of the E75G protein shows that the overall structure of mutant and WT proteins is similar. However, this mutation does cause a loss of essential hydrogen bonding and charge interactions between Glu(75) and Lys(9), Tyr(93), and His(121). In experiments using double point mutations, E73G/D77G, E73G/E75G, and E75G/D77G, significant changes are seen in all mutants in comparison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47, 59, and 58%, for E73G/D77G, E73G/E75G, and E75G/D77G, respectively. The triple mutant, E73G/E75G/D77G, results in fluorescence intensity and CD(222 nm) close to those of the denatured state and in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic interactions result in the formation of a locally stable structure in staphylococcal nuclease.     

Cooperativity network of Trp‐cage miniproteins: probing salt‐bridges

Trp‐cage miniprotein was used to investigate the role of a salt‐bridge (Asp⁹–Arg¹⁶) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side‐chains are also part of a dense interaction network composed of electrostatic, H‐bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt‐bridge with those of the salt‐bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp‐cage miniproteins is more complex than a simple two‐state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt‐bridge stabilization; in the miniprotein variants equipped with salt‐bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt‐bridge deleted mutants. In this molecular framework Arg¹⁶ stabilizes more than Asp⁹ does, because of its higher degree of 3D‐fold cooperation. In conclusion, the Xxx$^{9} \leftrightarrow$ Yyy¹⁶ salt‐bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions

Establishing a quantitative understanding of the determinants of affinity in protein–protein interactions remains challenging. For example, TEM‐1/β‐lactamase inhibitor protein (BLIP) and SHV‐1/BLIP are homologous β‐lactamase/β‐lactamase inhibitor protein complexes with disparate K_d values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV‐1, results in a 1000‐fold enhancement in binding affinity. In TEM‐1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV‐1 D104 does not in the wild type SHV‐1/BLIP co‐structure. Here, we present a 1.6 Å crystal structure of the SHV‐1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV‐1 D104 and BLIP K74 and a 400‐fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Dynamics and cooperativity of Trp-cage folding

In this study, multiple independent molecular dynamics (MD) simulations on Trp-cage folding were performed at 300, 325 and 375 K using generalized Born (GB) implicit solvent model. The orientational movement of the side-chain of Trp6 to form a hydrophobic core with 3₁₀-helix was observed. The breaking/formation of a salt bridge between Asp9 and Arg16 was proposed to be the prerequisite for Trp-cage folding/refolding. Our results demonstrate that the cooperation between the salt bridge and the Trp6 orientation leads to a stable tertiary structure of Trp-cage. Analyses on backbone concerted motions at different temperatures indicate that interactions between Trp6 and 3₁₀-helix & Pro18 and between Pro12 and Pro17 & Pro18 are weakened at 375 K but strengthened at lower temperatures, suggesting that they could be the potential driving force of hydrophobic collapse.     

Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins: solution structures of mouse CRYPTDIN-4 and (E15D)-CRYPTDIN-4

alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu(15) residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.     

Contribution of salt bridges near the surface of a protein to the conformational stability

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.     

The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A.

The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three-dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 3.5-5.6 (Tm, 62 +/- 2 degrees C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerstr?m, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19-30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.     

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.     

Structure of a protein G helix variant suggests the importance of helix propensity and helix dipole interactions in protein design

Six helix surface positions of protein G (Gbeta1) were redesigned using a computational protein design algorithm, resulting in the five fold mutant Gbeta1m2. Gbeta1m2 is well folded with a circular dichroism spectrum nearly identical to that of Gbeta1, and a melting temperature of 91 degrees C, approximately 6 degrees C higher than that of Gbeta1. The crystal structure of Gbeta1m2 was solved to 2.0 A resolution by molecular replacement. The absence of hydrogen bond or salt bridge interactions between the designed residues in Gbeta1m2 suggests that the increased stability of Gbeta1m2 is due to increased helix propensity and more favorable helix dipole interactions.     

Structural Insights into Conformational Stability of Wild-Type and Mutant β1-Adrenergic Receptor

Recent experiments to derive a thermally stable mutant of turkey beta-1-adrenergic receptor (β₁AR) have shown that a combination of six single point mutations resulted in a 20°C increase in thermal stability in mutant β₁AR. Here we have used the all-atom force-field energy function to calculate a stability score to detect stabilizing point mutations in G-protein coupled receptors. The calculated stability score shows good correlation with the measured thermal stability for 76 single point mutations and 22 multiple mutants in β₁AR. We have demonstrated that conformational sampling of the receptor for various mutants improve the prediction of thermal stability by 50%. Point mutations Y227A^5.58, V230A^5.61, and F338M^7.48 in the thermally stable mutant m23-β₁AR stabilizes key microdomains of the receptor in the inactive conformation. The Y227A^5.58 and V230A^5.61 mutations stabilize the ionic lock between R139^3.50 on transmembrane helix3 and E285^6.30 on transmembrane helix6. The mutation F338M^7.48 on TM7 alters the interaction of the conserved motif NPxxY(x)_5,6F with helix8 and hence modulates the interaction of TM2-TM7-helix8 microdomain. The D186-R317 salt bridge (in extracellular loops 2 and 3) is stabilized in the cyanopindolol-bound wild-type β₁AR, whereas the salt bridge between D184-R317 is preferred in the mutant m23. We propose that this could be the surrogate to a similar salt bridge found between the extracellular loop 2 and TM7 in β₂AR reported recently. We show that the binding energy difference between the inactive and active states is less in m23 compared to the wild-type, which explains the activation of m23 at higher norepinephrine concentration compared to the wild-type. Results from this work throw light into the mechanism behind stabilizing mutations. The computational scheme proposed in this work could be used to design stabilizing mutations for other G-protein coupled receptors.     

16 alpha-iodo-3,17 beta-estradiol: a stable ligand for estrogen receptor determinations in tissues with high 17 beta-hydroxysteroid dehydrogenase activity

Recently, the successful synthesis of radioiodinated 16 alpha-iodo-3,17 beta-estradiol-[125I] [125I]E2 was reported [1]. This new ligand has similar binding characteristics to the estrogen receptor (ER) [2-5] as the currently used tritium labeled estradiol [3H]E2. However, it offers several advantageous features: (a) high specific activity (theoretically 2,000 Ci/mmol) [1]; (b) minor problems with radioactive waste due to its short half life and (c) the possibility of simultaneous determination of ER and progesterone receptors (PgR) by double labeling with [125I]E2 and [3H]R5020 [6, 7]. As we are presently trying to determine ER and PgR in human placental cytosols we were interested in the stability of different labeled estrogens under the conditions of ER-assay. Placental cytosols [8] as well as cytosols of other tissues such as endometrium [9, 10], ovary [11] or mammary carcinomas [12] have been reported to contain significant amounts of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity. Conversion of labeled estradiol to estrone during incubation for ER-quantification would diminish the amount of labeled estradiol thus leading to errors in ER-concentrations, as estrone has only about 10% of estradiol's binding activity [13].     

Effects of engineered salt bridges on the stability of subtilisin BPN'

Variants designed using PROTEUS have been produced in an attempt to engineer stabilizing salt bridges into subtilisin BPN'. All the mutants constructed by site-directed mutagenesis were secreted by Bacillus subtilis, except L75K. Q19E, expressed as a single variant and also in a double variant, Q19E/Q271E, appears to form a stabilizing salt bridge based on X-ray crystal structure determination and differential scanning calorimeter measurements. Although the double mutant was found to be less thermodynamically stable than the wild-type, it did exhibit an autolytic stability about two-fold greater under hydrophobic conditions. Four variants, A98K, S89E, V26R and L235R, were found to be nearly identical to wild-type in thermal stability, indicative of stable structures without evidence of salt bridge formation. Variants Q271E, V51K and T164R led to structures that resulted in varying degrees of thermodynamic and autolytic instability. A computer-modeling analysis of the PROTEUS predictions reveals that the low percentage of salt bridge formation is probably due to an overly simplistic electrostatic model, which does not account for the geometry of the pairwise interactions.