Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags

Careful analysis of sub-visible amorphous aggregates, where proteins associate non-covalently in either native or denatured states without forming a specific quaternary structure, may shed insight into the mechanisms of protein aggregation and solubility. Here we report a biophysical and biochemical analysis of our model protein, a bovine pancreatic trypsin inhibitor variant (BPTI-19A), whose oligomerization were controlled by attaching solubility controlling peptide tags (SCP tags) to its C terminus, which are short peptides composed of a single type of amino acid that modulate protein solubility. The dynamic light scattering and static light scattering at 25 °C indicated that 11 out of 15 SCP tags merely affected the hydrodynamic radius and light scattering intensity of our reference variants BPTI-19A and BPTI-C2G. On the other hand, hydrophobic SCP tags composed of 5 Ile (C5I) or 5 Leu (C5L) were associated into sub-visible aggregates. Circular dichroism indicated that all tagged BPTI variants had the same secondary structure contents as the reference BPTI-19A at 25 °C, suggesting that BPTI-C5I and C5L kept their native structure upon association. Furthermore, the thermal denaturation of all of the BPTI variants was fully reversible and typical of natively folded small globular proteins, as monitored by CD at 222 nm. However, the thermal stability of BPTI-19A tagged with hydrophobic residues decreased with increasing protein concentration and tag's hydrophobicity, and BPTI-C5I and C5L were partially denatured at 37 °C. Biochemical stability assessed by limited proteolysis with pepsin correlated with the extent of the variants' aggregation, and the large sub-visible aggregates formed by BPTI-C5I and C5L significantly increased their resistance to pepsin proteolysis. Altogether, these observations indicated that hydrophobic SCP tags led to the reversible association of native-like proteins into sub-visible soluble amorphous aggregates resistant to pepsin digestion.     

Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20?^°C, ～85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15?mg which was ～3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37?^°C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.     

Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

A non-polar patch on the surface of a protein can cause a reduction in the solubility and stability of the protein, and thereby induce aggregation. However, a non-polar patch may be required so that the protein can bind to another molecule. The mutant 6L—derived from the acidic, dimeric α-helical protein sulerythrin and containing six additional leucines arranged to form a non-polar patch on its surface when properly folded—has a substantially reduced solubility in comparison with that of wild-type sulerythrin. This reduced solubility appears to cause 6L to aggregate. To reverse this aggregation, we mutated 6L so that it contained three to six additional glutamates or aspartates that we predicted would surround the non-polar leucine patch on natively folded 6L. Although the introduction of three glutamates or aspartates increased solubility, the mutants still aggregate and have a reduced α-helical content. Conversely, mutants with six additional glutamates or aspartates appear to exist mostly as dimers and to have the same α-helical content as that of wild-type sulerythrin. Notably, the introduction of five lysines or five arginines at the positions held by the glutamates or aspartates did not recover solubility as effectively as did the negatively charged residues. These results demonstrate that negatively charged residues, but not positively charged ones, surrounding a non-polar patch on an acidic protein can completely reverse the decrease in its solubility caused by the patch of non-polar surface residues.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.     

The formation of viroplasm-like structures by the rotavirus NSP5 protein is calcium regulated and directed by a C-terminal helical domain

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic alpha-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 alpha-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted alpha-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.     

Thermodynamic properties of BPTI variants with highly simplified amino acid sequences

We report the first detailed thermodynamic analysis of simplified proteins by differential scanning calorimetry (DSC). The experiments were carried out with five simplified BPTI variants, whose structures and activities have been reported, in which several residues not essential for specifying the tertiary structure were replaced by alanine. In most aspects, the thermodynamics of simplified proteins were very similar to, if not essentially identical with, those of natural proteins. In particular, they undergo a highly cooperative two-state thermal unfolding process with a large enthalpy change, which is a thermodynamic hallmark of the native state of natural globular proteins. Furthermore, the specific enthalpy and entropy changes upon unfolding at 110 degrees C were close to values invariably observed for small natural globular proteins (55 J g(-1) and ~16 J K(-1) g(-1), respectively). On the other hand, two simplified BPTI variants, BPTI-21 and BPTI-22 (containing 21 and 22 alanine residues), were enthalpically stabilized while entropically destabilized with respect to the reference BPTI-[5,55] molecule. This peculiar type of entropy-enthalpy compensation is in sharp contrast to the usual enthalpy destabilization/entropy stabilization observed in mutational studies of natural proteins. Overall, we conclude that a thermodynamic native state can be achieved by proteins encoded with extensively simplified sequences.     

Biophysical properties of gammaC-crystallin in human and mouse eye lens: the role of molecular dipoles

The eye lens is packed with soluble crystallin proteins, providing a lifetime of transparency and light refraction. γ-Crystallins are major components of the dense, high refractive index central regions of the lens and generally have high solubility, high stability and high levels of cysteine residues. Human γC belongs to a group of γ-crystallins with a pair of cysteine residues at positions 78 and 79. Unlike other γ-crystallins it has relatively low solubility, whereas mouse γC, which has the exposed C79 replaced with arginine, and a novel mouse splice variant, γCins, are both highly soluble. Furthermore, human γC is extremely stable, while the mouse orthologs are less stable. Evolutionary pressure may have favoured stability over solubility for human γC and the reverse for the orthologs in the mouse. Mutation of C79 to R79, in human γC, greatly increased solubility, however, neither form produced crystals. Remarkably, when the human γD R36S crystallization cataract mutation was mimicked in human γC-crystallin, the solubility of γC was dramatically increased, although it still did not crystallize. The highly soluble mouse γC-crystallin did crystallize. Its X-ray structure was solved and used in homology modelling of human γC, and its mutants C79R and R36S. The human γD R36S mutant was also modelled from human γD coordinates. Molecular dynamics simulation of the six molecules in the solution state showed that the human γCs differed from γDs in domain pairing, behaviour that correlates with interface sequence changes. When the fluctuations of the calculated molecular dipoles, for the six structures, over time were analysed, characteristic patterns for soluble γC and γD proteins were observed. Individual sequence changes that increase or decrease solubility correlated well with changes in the magnitude and direction of these dipoles. It is suggested that changes in surface residues have allowed adaptation for the differing needs of human and mouse lenses.     

Human influenza virus hemagglutinin with high sensitivity to proteolytic activation.

To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.     

Three consecutive arginines are important for the mycobacterial peptide deformylase enzyme activity

Genes encoding the peptide deformylase enzyme (def) are present in all eubacteria and are involved in the deformylation of the N-formyl group of newly synthesized polypeptides during protein synthesis. We compared the amino acid sequences of this enzyme in different mycobacterial species and found that they are highly conserved (76% homology with 62% identity); however, when this comparison was extended to other eubacterial homologs, it emerged that the mycobacterial proteins have an insertion region containing three consecutive arginine residues (residues 77-79 in Mycobacterium tuberculosis peptide deformylase (mPDF)). Here, we demonstrate that these three arginines are important for the activity of mPDF. Circular dichroism studies of wild-type mPDF and of mPDF containing individual conservative substitutions (R77K, R78K, or R79K) or combined substitutions incorporated into a triple mutant (R77K/R78K/R79K) indicate that such mutations cause mPDF to undergo structural alterations. Molecular modeling of mPDF suggests that the three arginines are distal to the active site. Molecular dynamics simulations of wild-type and mutant mPDF structures indicate that the arginines may be involved in the stabilization of substrate binding pocket residues for their proper interaction with peptide(s). Treatment with 5'-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against different regions of def from M. tuberculosis inhibits growth of Mycobacterium smegmatis in culture. Taken together, these results hold out the possibility of future design of novel mycobacteria-specific PDF inhibitors.     

Targeting of a tail-anchored protein to endoplasmic reticulum and mitochondrial outer membrane by independent but competing pathways

Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b(5), a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.     

Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein

PKI-(5-24)-amide is a 20-residue peptide with the sequence, Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-A la-Ile-His- Asp24-NH2, that corresponds to the active portion of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992). Amino acid residues in PKI-(5-24)-amide responsible for the potent inhibition (Ki = 2.3 nM) of the catalytic subunit of protein kinase were further investigated using deletion and substitution analogs of the synthetic peptide. Residues 5, 23, and 24 were not required for activity since the 17-residue PKI-(6-22)-amide retained full potency. Sequential removal of the first seven amino acids from the NH2 terminus of PKI-(5-24)-amide caused a progressive 50-fold loss of inhibitory potency. In contrast, substitution of either Thr6, Asp9, or Ile11 with alanine, or Ala8 by leucine, in PKI-(5-22)-amide produced less than 3-fold decreases in potency. Of the 2 aromatic residues in PKI-(5-22)-amide, the individual substitution of Phe10 and Tyr7 by alanine caused, respectively, 90- and 5-fold decreases in inhibitory potency, demonstrating important roles for each. This NH2-terminal portion of the peptide is believed to contain a significant portion of alpha-helix. Many recognition or structural determinants are also essential in the COOH-terminal portion of PKI-(5-22)-amide. In addition to the basic subsite provided by the three arginines, several other of the residues are critical for full inhibitory potency. Substitution of Ile22 by glycine in either PKI-(5-22)-amide or PKI-(14-22)-amide lowered the inhibitory potency by 150- and 50-fold, respectively. Separate replacement of Gly17 or Asn20, in either PKI-(5-22)-amide or PKI-(14-22)-amide, caused 7-15-fold decreases in potency. Substitution of both Gly17 and Asn20 together (in PKI-(14-22)-amide) produced a synergistic loss of inhibitory activity. [Leu13,Ile14]PKI-(5-22)-amide, a doubly substituted analog exhibited a 42-fold increase in Ki value. We conclude that Ser13 and/or Gly14, Gly17, Asn20, and Ile22 each contribute important features to the binding of these inhibitory peptides to the protein kinase, either by providing recognition determinants, inducing structure, and/or allowing essential peptide backbone flexibility.(ABSTRACT TRUNCATED AT 400 WORDS)     

Process intensification for the removal of poly‐histidine fusion tags from recombinant proteins by an exopeptidase

This study describes the use of a hexa‐histidine tagged exopeptidase for the cleavage of hexa‐histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On‐column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on‐column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post‐load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post‐load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa‐histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa‐histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on‐column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on‐column exopeptidase cleavage makes the integration of the poly‐histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly‐histidine tag removal can now be combined with the on‐column tag cleavage operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Acyl chain specificity of ceramide synthases is determined within a region of 150 residues in the Tram-Lag-CLN8 (TLC) domain

In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl-CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and -5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N terminus) and half of CerS2 (from the C terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 toward C16-CoA; likewise, the putative sixth transmembrane domain (at the C terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity toward C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity toward C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity.     

Tailoring of recombinant FDH: effect of histidine tag location on solubility and catalytic properties of Chaetomium thermophilum formate dehydrogenase (CtFDH)

Abstract

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD⁺ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD⁺ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.     

In silico study on the effect of surface lysines and arginines on the electrostatic interactions and protein stability

Charged amino acids are mostly exposed on a protein surface, thereby forming a network of interactions with the surrounding amino acids as well as with water. In particular, positively charged arginine and lysine have different side chain geometries and provide a different number of potential electrostatic interactions. This study reports a comparative analysis of the difference in the number of two representative electrostatic interactions, such as salt-bridges and hydrogen bonds, contributed by surface arginine and lysine, as well as their effect on protein stability using molecular modeling and dynamics simulation techniques. Two in silico variants, the R variant with all arginines and the K variant with all lysines on the protein surface, were modeled by mutating all the surface lysines to arginines and the surface arginines to lysines, respectively, for each of the 10 model proteins. A structural comparison of the respective two variants showed that the majority of R variants possessed more salt-bridges and hydrogen bond interactions than the K variants, indicating that arginine provides a higher probability of electrostatic interactions than lysine owing to its side chain geometry. Molecular dynamics simulations of these variants revealed the R variants to be more stable than the K variants at room temperature but this effect was not prominent under protein denaturating conditions, such as 353 and 333 K with 8 M urea. These results suggest that the arginine residues on a protein surface contribute to the protein stability slightly more than lysine by enhancing the electrostatic interactions.     

Identification by mutagenesis of conserved arginine and tryptophan residues in rat liver carnitine palmitoyltransferase I important for catalytic activity

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.     

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.