Effects of Mutations of Lys41 and Asp102 of Bacteriorhodopsin

Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK _a of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK _a of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.     

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.     

Connectivity of the retinal Schiff base to Asp85 and Asp96 during the bacteriorhodopsin photocycle: the local-access model.

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.     

β-Turn structure and intramolecular interaction of tetrapeptides containing Asp and Lys

Nmr and CD studies of terminally protected tetrapeptides were carried out in aqueous and DMSO solutions to investigate the formation and stabilization of the β-turn structure. Boc-Gly-Lys-Asp-Gly-OMe and Boc-Asp-Lys-Asp-Gly-OMe appear to have a tendency to adopt a β-turn structure in aqueous solution from the CD spectra and temperature-dependence studies of the amide proton chemical shifts. The side-chain conformation of the Asp residue depends greatly on its ionization state but was not affected by the deprotonation of the neighboring Lys side chain. There is evidence for an intramolecular interaction between the Asp and Lys side chains of Boc-Gly-Lys-Asp-Gly-OMe. Such an interaction can contribute to the stabilization of the β-turn structure.     

Site-directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin

The functional contributions of amino acid residues Thr218 and Asp304 of chymosin, both of which are highly conserved in the aspartic proteinases, are analysed by means of site-directed mutagenesis. The optimum pH values, milk-clotting (C) and proteolytic (P) activities and kinetic parameters for synthetic oligopeptides as substrates were examined for the mutant enzymes. The mutation Thr218Ser caused a marked increase in the C/P ratio, which seemed to be due to a change in substrate recognition. Although the negative charge of Asp304 had been expected to play a role in lowering the optimum pH values in the aspartic proteinases, this turned out not to be the case in chymosin because both the mutations Asp304Ala and Asp304Glu caused a similar shift of the optimum pH towards the acidic side. In addition, the mutation Lys220Leu, which we generated previously, was found to cause a decrease in the C/P ratio, mainly due to the increase in the proteolytic activity.     

Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions.

The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->Asn and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, both in the presence of Cl- (150 mM) and in its absence (75 mM SO4(2-)). Under both conditions the color of the pigment is blue (lambda max = 615 nm). The time course of the photovoltage kinetics is similar to that of the acid-blue form of wild-type, except that an additional transient charge motion occurs with time constants of 60 microseconds and 1.3 ms, indicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is suggested that this is the group XH, which is responsible for proton release in wild-type. At pH 1, the photovoltage signal of D85N changes upon the addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore, the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pKa of the Schiff base, there is a mixture of "M-like" and "N-like" states. Net proton transport in the same direction as in wild-type is restored in D85N from this N-like state. With the mutant Asp212-->Asn (D212N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire pH range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage experiments indicate that net proton pumping is restored. However, this Cl(-)-induced activation of net charge-transport in D212N is only partial. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chloride-dependent features as those accompanying the acid-blue to acid-purple transition of the wild-type.     

Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

The function of Asp70, Glu77 and Lys79 in the Escherichia coli MutH protein

The MutH protein, which is part of the Dam-directed mismatch repair system of Escherichia coli, introduces nicks in the unmethylated strand of a hemi-methylated DNA duplex. The latent endonuclease activity of MutH is activated by interaction with MutL, another member of the repair system. The crystal structure of MutH suggested that the active site residues include Asp70, Glu77 and Lys79, which are located at the bottom of a cleft where DNA binding probably occurs. We mutated these residues to alanines and found that the mutant proteins were unable to complement a chromosomal mutH deletion. The purified mutant proteins were able to bind to DNA with a hemi-methylated GATC sequence but had no detectable endonuclease activity with or without MutL. Although the data are consistent with the prediction of a catalytic role for Asp70, Glu77 and Lys79, it cannot be excluded that they are also involved in binding to MutL.     

An alpha 1-antitrypsin variant, Pi B Alhambra (Lys to Asp, Glu to Asp), with rapid anodal electrophoretic mobility.

A variant of human alpha 1-antitrypsin (alpha 1 AT) was found by acid starch gel electrophoresis and by thin-layer electrofocusing. The variant has an anodal migration velocity almost identical to PiB. It is designated as Pi B Alhambra. Pi B Alhambra was purified to homogeneity from a heterozygous PiM1/PiB Alhambra subject. Specific trypsin inhibitory activity and composition of amino acids and carbohydrates were similar to those of normal PiM1. The structural difference between the normal and the variant inhibitors was elucidated by peptide mapping of their tryptic digests. Two amino acid substitutions, Lys to Asp and Glu to Asp, were found. The amino acid substitution, Gly to Asp, has been found in a common PiM2 variant [1]. The Pi B Alhambra variant presumably originated by two steps of mutation: generation of PiM2 from wild type PiM1 by the substitution Gly to Asp, and subsequent generation of Pi B Alhambra from PiM2 by another substitution, Lys to Asp.     

A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Addition of azide fully restored the proton pump activity of defective bacteriorhodopsin (BR) mutant protein Asp96----Asn. The decay time of M of BR Asp96----Asn, the longest living intermediate, was decreased from 500 ms at pH 7.0 to approximately 1 ms under conditions of saturating azide concentrations. This decay was faster than the decay of M in the wild-type, where no such azide effect was detectable. Stationary photocurrents, measured with purple membranes immobilized and oriented in a polyacrylamide gel, increased upon addition of azide up to the level of the wild-type. Different small anions of weak acids restored the pump activity with decreasing affinity in the order: cyanate greater than azide greater than nitrite greater than formiate greater than acetate. The activation energy of the M decay in the mutant was higher in the presence (48 kJ/mol) than in the absence (27 kJ/mol) of 100 mM azide even though the absolute rate was dramatically increased by azide. This effect of azide is due to the substitution of a carboxamido group for a carboxylic group at position 96 which removes the internal proton donor and causes an increase in the entropy change of activation for proton transfer which is reversed by azide.     

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia

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Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.

Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.     

Lys 43 and Asp 46 in alpha-helix 3 of uteroglobin are essential for its phospholipase A2 inhibitory activity

Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A2 (sPLA2)-inhibitory activity. However, the mechanism by which UG inhibits sPLA2 activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four alpha-helices. We previously reported that sPLA2-inhibitory activity of UG may reside in a segment of alpha-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA2 activity by binding and sequestering Ca++, essential for sPLA2 activation. By site-specific mutation, we demonstrate here that Lys 43 Glu, Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA2-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca++ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca++. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA2-inhibitory activity of UG and (ii) Ca++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA2-inhibitory activity.     

Thermostability of the HIV gp41 wild-type and loop mutations

The HIV and SIV gp41 ectodomains are extremely stable to chemical and thermal denaturation and the observed stability has been proposed to be an important thermodynamic driving force for gp41-mediated fusion of the viral and target cell membranes. The importance of the disulphide bond and surrounding residues within the HIV gp41 loop have been assayed by DSC studies of wild type and mutant HIV gp41. Based on the thermal transition temperature, the disulphide bond and surrounding residues do not contribute to the thermal stability of gp41 and thus do not contribute to gp41-mediated membrane fusion.     

Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase

Thr(373), Lys(374), Asp(375), and Lys(260) were chosen as site-directed mutagenesis targets within porcine NADP-dependent isocitrate dehydrogenase based on structurally corrected sequence alignment among prokaryotic and eukaryotic NADP-isocitrate dehydrogenases. Wild-type and all mutant enzymes were expressed in Escherichia coli and purified to homogeneity. These mutations do not alter the secondary structure or dimerization state of the mutants. The D375N and K260Q mutants exhibit, respectively, a 15- and 28-fold increase in K(m) for NADP, along with marked decreases in V(max) as compared to wild-type enzyme. In contrast, replacing Lys(374), which was previously proposed to contribute to apparent coenzyme affinity, does not change the enzyme's kinetic parameters. T373S exhibits similar kinetic parameters to those of wild-type while T373A and T373V mutations reduce the V(max) values of the resulting enzymes to 1 and 20%, respectively of that of wild-type. We conclude that a hydroxyl group at position 373 is required for effective enzyme function and that Asp(375) and Lys(260) are critical amino acids contributing to coenzyme affinity as well as catalysis by porcine NADP-isocitrate dehydrogenase.     

The mutations Lys 114 --> Gln and Asp 126 --> Asn disrupt an intersubunit salt bridge and convert Listeria innocua Dps into its natural mutant Listeria monocytogenes Dps. Effects on protein stability at Low pH

The stability of the dodecameric Listeria monocytogenes Dps has been compared with that of the Listeria innocua protein. The two proteins differ only in two amino acid residues that form an intersubunit salt-bridge in L. innocua Dps. This salt-bridge is replaced by a hydrogen bonding network in L. monocytogenes Dps as revealed by the X-ray crystal structure. The resistance to low pH and high temperature was assayed for both Dps proteins under equilibrium conditions and kinetically. Despite the identical equilibrium behavior, significant differences in the kinetic stability and activation energy of the unfolding process are apparent at pH 1.5. The higher stability of L. monocytogenes Dps has been accounted for in terms of the persistence of the hydrogen bonding network at this low pH value. In contrast, the salt-bridge between Lys 114 and Asp 126 characteristic of L. innocua Dps is most likely abolished due to protonation of Asp 126.     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.     

Hydrolysis of chimeric proteins by enteropeptidase at the specific linker (Asp)4Lys depending on refolding conditions

Refolding from inclusion bodies of chimeric proteins containing the enteropeptidase-specific linker (Asp)4Lys was carried out. It was shown that, depending on the refolding conditions, chimeric proteins function as substrates or inhibitors of the enteropeptidase. The efficiency of the enteropeptidase hydrolysis of chimeric proteins containing the (Asp)4Lys linker may depend not only on the amino acid sequence of the protein binding site for the enzyme but also on the site conformation.     

Structure-function studies of bacteriorhodopsin XV. Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure

Bacteriorhodopsin mutants containing deletions in loop B-C, delta Thr67-Glu74 or delta Gly65-Gln75 or a deletion in the loop E-F, delta Glu161-Ala168, were prepared. Following their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures. The mutants containing deletions in the loop B-C were normal at 4 degrees C but showed the following changes at 20 degrees C. 1) The lambda max shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased. Deletion of the eight amino acids in loop E-F did not affect wild-type behavior. However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild-type bacteriorhodopsin. These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they contribute to the stability of the folded protein.     

Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein

We have studied the cleavage efficiency of the protease enterokinase (EK) using the novel vector pESP4. pESP4 is a yeast expression vector equipped with ligation-independent cloning sites, a GST purification tag, and a FLAG epitope tag. EK is used to cleave the FLAG and GST tags leaving the protein of interest without any extraneously added amino acids. We have found that EK is relatively permissive of the amino acid residue downstream of the recognition sequence (the P'1 position). This makes EK an ideal choice to use as a protease to cleave any protein of interest cloned within the pESP4 yeast expression vector.