Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.     

Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumors

An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 +/- 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 +/- 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using (124)I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.     

Isolation and characterization of mutants of firefly luciferase which produce different colors of light

The luciferase cDNA from the ‘Genji’ firefly, Luciolacruciata, was mutated with hydroxylamine to isolate mutant lucierases.Some of the isolated mutant enzymes produced different colorsof light, ranging from green to red. Five such mutants, producinggreen (_max = 558 nm), yellow-orange (_max = 595 nm), orange (_max= 607 nm) and red light (_max = 609 and 612 nm), were analyzed.The mutations were found to be single amino acid changes, fromVal239 to IIe, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser andHis433 to Tyr respectively.     

Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease

To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.     

Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs

Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.     

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

Variants designed using PROTEUS have been produced in an attemptto engineer stabilizing salt bridges into subtilisin BPN'. Allthe mutants constructed by site-directed mutagenesis were secretedby Bacillus subtillus, except L75K. Q19E, expressed as a singlevariant and also in a double variant, Q19E/Q271E, appears toform a stabilizing salt bridge based on X-ray crystal structuredetermination and differential scanning calorimeter measurements.Although the double mutant was found to be less thermodynamicallystable than the wild-type, it did exhibit an autolytic stabilityabout two fold greater under hydrophobic conditions. Four variants,A98K, S89E, V26R and L235R, were found to be nearly identicalto wild-type in thermal stability, indicative of stable structureswithout evidence of salt bridge formation. Variants Q271E, V51Kand T164R led to structures that resulted in varying degreesof thermodynamic and autolytic instability. A computer-modelinganalysis of the PROTEUS predictions reveals that the low percentageof salt bridge formation is probably due to an overly simplisticelectrostatic model, which does not account for the geometryof the pairwise interactions.     

Role of Arg163 in the N-glycosidase activity of neo-trichosanthin

Three mutant crystals of neo-trichosanthin (n-TCS), R163K, R163Hand R163Q, were obtained by the hanging drop vapor diffusionmethod. Structure determination indicated that there are nosignificant differences between the mutants and n-TCS exceptin the active pocket. All of them were also soaked in sodiumcitrate buffer (pH 4.5) containing 20% KCl and 10 mg/ml AMP.Structure determination suggests that in the active pocket ofthe crystals of R163K and R163H, parallel to the aromatic ringof Tyr70, each mutant possesses an adenine. The relationshipbetween structure and function is discussed. Biochemical analysisreveals that the mutants R163K and R163H have N-glycosidaseactivity, while R163Q does not. This suggests that R163 is acrucial residue for the enzyme activity of n-TCS, and its roleis providing proton.     

The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability

The net energetic contribution of interhelical electrostaticattractions to coiled-coil stability has been quantitated usingde novo designed synthetic coiled-coils. The synthesized modelcoiled-coil (EK), denoted by amino acid res-idues in positionse and g, which contains only interhelical ionic interactionswithout any possible (i, i + 3) and (i, i + 4) intrahelicalionic interaction, consists of two identical 35 residue polypeptidechains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coilswere prepared where five Glu residues at e positions in EK weremutated to Gin residues (QK); five Lys residues at g positionswere altered to Gin residues (EQ) or these mutations were effectedat both positions e and g (QQ). The stabilities of the fourcoiled-coils were determined by measuring the ellipticitiesat 220 nm as a function of urea concentration at 20C. By usinga double-mutant cycle analysis it was possible to isolate theenergetic contribution of interhelical ionic attractions tocoiled-coil stability from the other contributions such as helicalpreference and hydro-phobicity. The 0.37 0.01 kcal/mol ofenergetic contribution of one interhelical ion pair to the coiled-coilstability was obtained from three independent comparisons. Thisfinding suggests that a large number of weak interhelical electrostaticinteractions on the surface of a protein can make a substantialcontribution to protein stability. In addition, the energeticcontributions of a single mutation E^– Q, K⁺Q, Q E andE^– Ewere also determined (G = 0.22, 0.26, 0.46 and 0.65kcal/mol for the single mutations, respectively). The greatercontribution of a protonated Glu residue to coiled-coil stabilitycompared with an ionized Glu residue (0.65 kcal/mol) can outweighthe relatively smaller contribution of an interhelical ion pair(0.37 kcal/mol), which clearly explains why most coiled-coilsare more stable at acidic pH compared with neutral pH even wheninterhelical salt bridges contribute to the coiled-coil stabilityat neutral pH.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability

To achieve a thermostable beta-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70 degrees C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80 degrees C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100 degrees C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.     

Red fluorescent protein variants with incorporated non-natural amino acid analogues

Fluorescent proteins are important tools in biotechnology applications and biosensing. DsRed, a red fluorescent protein, has expanded the colors of fluorescent proteins beyond the more commonly used green fluorescent protein. Many genetic modifications have been performed on DsRed to overcome some of its drawbacks. These primarily focused on overcoming the oligomerization detrimental to DsRed activity, and the parasitic green fluorescence caused by the immature chromophore. One such variant, DsRed-monomer, has minimal green fluorescence and no oligomerization. A few traditional mutagenesis studies have been done with DsRed and its mutants to shift the fluorescence wavelengths creating additions to the pallet of fluorescent protein colors. We have explored incorporation of non-natural amino acid analogues into DsRed-Monomer, obtaining variants with differing emission properties. In this work, two such analogues of tyrosine have been incorporated into DsRed-Monomer: 3-amino-l-tyrosine and 3-fluoro-l-tyrosine. Tyrosine analogues were chosen due to the role of tyrosine in the formation and structure of the protein's chromophore. The variants obtained in our study showed altered emission wavelengths and spectral characteristics. Our study demonstrates that incorporation of non-natural analogues into DsRed-Monomer is a viable approach to alter the spectral characteristics of the protein. We envision that this study will open up the door to non-natural mutagenesis studies with red fluorescent proteins and its mutants.     

Different disease-causing mutations in transthyretin trigger the same conformational conversion

Transthyretin (TTR)-containing amyloid fibrils are deposited in cardiac tissue as a natural consequence of aging. A large number of inherited mutations lead to amyloid diseases by accelerating TTR deposition in other organs. Amyloid formation is preceded by a disruption of the quaternary structure of TTR and conformational changes in the monomer. To study conformational changes preceding the formation of amyloid, we performed molecular dynamics simulations of the wild-type monomer, amyloidogenic variants (V30M, L55P, V122I) and a protective variant (T119M) at neutral and low pH. At low pH, the D strand dissociated from the beta-sheet to expose the A strand, consistent with experimental studies. In amyloidogenic variants and in the wild-type at low pH, there was a conformational change in the beta-sheets into alpha-sheet via peptide bond flips that was not observed at neutral pH in the wild-type monomer. The same residues participated in conversion in each amyloidogenic variant simulation, originating in the G strand between residues 106 and 109, with accelerated conversion at low pH. The T119M protective variant changed the local conformation of the H strand and suppressed the conversion observed in amyloidogenic variants.     

Construction of stabilized proteins by combinatorial consensus mutagenesis

We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E.cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and eight mutations that destabilized the protein. A second-generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees C higher than the starting molecule and contained eight consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.     

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging

New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin–luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.     

Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive

A number of mutations have been shown previously to stabilizeT4 lysozyme. By combining up to seven such mutations in thesame protein, the melting temperature was incrementally increasedby up to 83°C at pH 5.4 (G = 3.6 kcal/mol). This shows thatit is possible to engineer a protein of enhanced thermostabilityby combining a series of rationally designed point mutations.It is also shown that this stabilization is achieved with onlyminor, localized changes in the structure of the protein. Thisis consistent with the observation that the change in stabilityof each of the multiple mutants is, in each case, additive,i.e. equal to the sum of the stability changes associated withthe constituent single mutants. One of the seven substitutions,Asn116 Asp, changes a residue that participates in substratebinding; not surprisingly, it causes a significant loss in activity.Ignoring this mutation, there is a gradual reduction in activityas successively more mutations are combined.     

Molecular mechanics analysis of drug-resistant mutants of HIV protease

Drug-resistant mutants of HIV-1 protease limit the long-termeffectiveness of current anti-viral therapy. In order to studydrug resistance, the wild-type HIV-1 protease and the mutantsR8Q, V32I, M46I, V82A, V82I, V82F, I84V, V32I/I84V and M46I/I84Vwere modeled with the inhibitors saquinavir and indinavir usingthe program AMMP. A new screen term was introduced to reproducemore correctly the electron distribution of atoms. The atomicpartial charge was represented as a delocalized charge distributioninstead of a point charge. The calculated protease–saquinavirinteraction energies showed the highly significant correlationof 0.79 with free energy differences derived from the measuredinhibition constants for all 10 models. Three different protonationstates of indinavir were evaluated. The best indinavir modelincluded a sulfate and gave a correlation coefficient of 0.68between the calculated interaction energies and free energiesfrom inhibition constants for nine models. The exception wasR8Q with indinavir, probably due to differences in the solvationenergy. No significant correlation was found using the standardmolecular mechanics terms. The incorporation of the new screencorrection resulted in better prediction of the effects of inhibitorson resistant protease variants and has potential for selectingmore effective inhibitors for resistant virus.     

Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis

We undertook a structure–function analysis of human tissueplasminogen activator (tPA) using linker-scanning and deletionmutagenesis. Synthetic oligonucleotide linkers were introducedinto the tPA cDNA at pre-existing restriction enzyme sites.This generated a series of tPA variants which contained smallprimary sequence alterations consisting of point mutations,deletions or insertions. The majority of the linker-insertionvariants demonstrate a significant reduction in amidolytic andfibrinolytic activity in comparison to wild-type tPA. The exceptionsare the variants with linker-inserts placed at the BglII(115)and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively),which encode insertions at the boundaries of the finger domain.The variants with linker-inserts in the light chain (proteasedomain) of tPA are the lowest in enzymatic activity. Particularlysensitive to mutation are highly conserved amino acids. Heavychain deletion variants were constructed from point mutantsat the domain boundaries of tPA. Deletion of the kringle domainslowers the fibrinolytic activity to a greater extent than deletionof the finger or growth factor domains. We conclude that alterationsin any domain of the tPA molecule, and particularly in the highlyconserved residues within these domains, can affect fibrinolyticactivity.     

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a verytight and rigid dimer interface. At this interface a criticalhydrogen bond is formed between the main chain oxygen atom ofthe catalytic residue Lys13 and the completely buried side chainof Gln65 (of the same subunit). The sequence of Leishmania mexicanaTIM, closely related to Trypanosoma brucei TIM (68% sequenceidentity), shows that this highly conserved glutamine has beenreplaced by a glutamate. Therefore, the 1.8 Å crystalstructure of leishmania TIM (at pH 5.9) was determined. Thecomparison with the structure of trypanosomal TIM shows no rearrangementsin the vicinity of Glu65, suggesting that its side chain isprotonated and is hydrogen bonded to the main chain oxygen ofLys13. Ionization of this glutamic acid side chain causes apH-dependent decrease in the thermal stability of leishmaniaTIM. The presence of this glutamate, also in its protonatedstate, disrupts to some extent the conserved hydrogen bond network,as seen in all other TIMs. Restoration of the hydrogen bondingnetwork by its mutation to glutamine in the E65Q variant ofleishmania TIM results in much higher stability; for example,at pH 7, the apparent melting temperature increases by 26°C(57°C for leishmania TIM to 83°C for the E65Q variant).This mutation does not affect the kinetic properties, showingthat even point mutations can convert a mesophilic enzyme intoa superstable enzyme without losing catalytic power at the mesophilictemperature.     

Structural rearrangements near the chromophore influence the maturation speed and brightness of DsRed variants

The red fluorescent protein DsRed has been extensively engineered for use as an in vivo research tool. In fast maturing DsRed variants, the chromophore maturation half-time is approximately 40 min, compared to approximately 12 h for wild-type DsRed. Further, DsRed has been converted from a tetramer into a monomer, a task that entailed mutating approximately 20% of the amino acids. These engineered variants of DsRed have proven extremely valuable for biomedical research, but the structural basis for the improved characteristics has not been thoroughly investigated. Here we present a 1.7 A crystal structure of the fast maturing tetrameric variant DsRed.T4. We also present a biochemical characterization and 1.6 A crystal structure of the monomeric variant DsRed.M1, also known as DsRed-Monomer. Analysis of the crystal structures suggests that rearrangements of Ser69 and Glu215 contribute to fast maturation, and that positioning of the Lys70 side chain modulates fluorescence quantum yield. Despite the 45 mutations in DsRed.M1 relative to wild-type DsRed, there is a root-mean-square deviation of only 0.3 A between the two structures. We propose that novel intramolecular interactions in DsRed.M1 partially compensate for the loss of intermolecular interactions found in the tetramer.     

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.