We report on comparative pre‐steady‐state kinetic analyses of exonuclease‐deficient Escherichia coli DNA polymerase I (Klenow fragment, KF − ) and the archaeal Y‐family DinB homologue (Dbh) of Sulfolobus solfataricus . We used size‐augmented sugar‐modified thymidine‐5′‐triphosphate (T R TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4′‐position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40‐fold for KF − and about twelvefold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF − for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T Me TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF − for these nucleotides drop by about 16‐ and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size‐augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.

The site-selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein-modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C-terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. with the exception of the biotin-functionalized analogue, all the phos-phoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)-8-(azidoacetamido)-3,7-dimethylocta-2,6-dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diets-Alder cycloaddition with 6-maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels-Alder cycloaddition. Our data validate the use of FTase as a protein-modification tool for biochemical and biotechnological applications.

AbstractThe site‐selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein‐modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C‐terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. With the exception of the biotin‐functionalized analogue, all the phosphoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)‐8‐(azidoacetamido)‐3,7‐dimethylocta‐2,6‐dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diels–Alder cycloaddition with 6‐maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels–Alder cycloaddition. Our data validate the use of FTase as a protein‐modification tool for biochemical and biotechnological applications.

Modification of proteins with polymers is a viable method to tune protein properties, e. g., to render them more water-soluble by using hydrophilic polymers. We have utilized precision- length, polyethylene glycol-based oligomers carrying a thioester moiety in transthioesterification and native chemical ligation reactions with internal and N-terminal cysteine residues in proteins and peptides. These reactions lead to uniquely modified proteins with an increased solubility in chaotrope- and detergent-free aqueous systems. Polymer modification of internal cysteines is fully reversible and allows generation of stable protein-polymer conjugates for enzymatic manipulations as demonstrated by proteolytic cleavage of a protein construct that was only soluble in buffers incompatible with protease activity before polymer modification. The permanent polymer modification of a Rab protein at its N-terminal cysteine produced a fully active Rab variant that was efficiently prenylated. Thus, PEGylation of prenylated proteins might be a viable route to increase water solubility of such proteins in order to carry out experiments in detergent- and lipid-free systems.

The development of the Diels-Alder cycloaddition as a new method for the site-specific chemoselective ligation of peptides and proteins under mild conditions is reported. Peptides equipped with a 2,4-hexadienyl ester and an N-terminal maleimide react in aqueous media to give cycloadducts in high yields and depending on the amino acid sequence with high stereoselectivity. Except for the cysteine SH group the transformation is compatible with all amino acid side chain functional groups. For ligation to proteins the hexadienyl group was attached to avidin and streptavidin noncovalently by means of complex formation with a biotinylated peptide or by covalent attachment of a hexadienyl ester-containing label to lysine side chains incorporated into the proteins. Site-specific attachment of the hexadienyl unit into a Rab protein was achieved by means of expressed protein ligation followed by protection of the generated cysteine SH by means of Ellman's reagent. The protein reacted with different maleimido-modified peptides under mild conditions to give the fully functional cycloadducts in high yield. The results demonstrate that the Diels-Alder ligation offers an advantageous and technically straightforward new opportunity for the site-specific equipment of peptides and proteins with further functional groups and labels. It proceeds under very mild conditions and is compatible with most functional groups found in proteins. Its combination with other ligation methods, in particular expressed protein ligation is feasible.

Here we report a detailed characterization of the biochemical and kinetic properties of the hepatitis C virus (HCV, genotype-1b, J4 consensus) RNA-dependent RNA polymerase NS5B, by performing comprehensive RNA binding, nucleotide incorporation, and protein/protein oligomerization studies. By applying equilibrium fluorescence titrations, we determined a surprisingly high dissociation constant (K(d)) of approximately 250 nM for single-stranded as well as for partially double-stranded RNA. A detailed analysis of the nucleic acid binding mechanism using pre-steady-state techniques revealed the association reaction to be nearly diffusion controlled. It occurs in a single step with a second-order rate constant (k(on)) of 0.273 nM(-)(1) s(-)(1). The dissociation of the nucleic acid-polymerase complex is fast with a dissociation rate constant (k(off)) of 59.3 s(-)(1). With short, partially double-stranded RNAs, no nucleotide incorporation could be observed, while de novo RNA synthesis with short RNA templates showed nucleotide incorporation and end-to-end template switching events. Single-turnover, single-nucleotide incorporation studies (representing here the initiation and not processive polymerization) using dinucleotide primers revealed a very slow incorporation rate (k(pol)) of 0.0007 s(-)(1) and a K(d) of the binary enzyme-nucleic acid complex for the incoming ATP of 27.7 microM. Using dynamic laser light scattering, it could be shown for the first time that oligomerization of HCV NS5B is a dynamic and monovalent salt concentration dependent process. While NS5B is highly oligomeric at low salt concentrations, monomers were only observed at NaCl concentrations above 300 mM. Binding of short RNA substrates led to a further increase in oligomerization, whereas GTP did not show any effect on protein/protein interactions. Furthermore, nucleotide incorporation studies indicate the oligomerization state does not correlate with enzymatic activities as previously proposed.

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nM, which is similar to 10- fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP ( at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity ( K-d similar to 600 mu M), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation ( correct and incorrect) is the chemical step ( phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.

Mild attachment: Proteins can be ligated chemoselectively and immobilized on functionalized glass surfaces by means of Diels–Alder cycloadditions (see scheme).

To examine the concept of polymerase active site tightness as a criteria for DNA polymerase fidelity, we performed pre-steady-state single nucleotide incorporation kinetic analyses with sugar modified thymidine 5′-triphosphate (TTP) analogues and human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The employed TTP analogues (TRTP) are modified at the 4′-position of the sugar moiety with alkyl groups, gradually expanding their steric demand. Introduction of a methyl group reduces the maximum rate of nucleotide incorporation by about 200-fold for RTWT and about 400-fold for RTM184V. Interestingly, the affinity of RT for the modified nucleotide is only marginally affected. Increasing the size to an ethyl group leads to further reduction of the rate of incorporation and first effects on binding affinities are observed. Finally, substitution for an isopropyl group results not only in a further reduction of incorporation rates but also in a dramatic loss of binding affinity for the nucleotide analogue. By increasing the steric demand the effects on RTM184V in comparison with RTWT become progressively more pronounced. Misincorporation of either TTP or TMeTP opposite a template G causes additional decline in incorporation rates accompanied by a drastic decrease in binding affinities. This results in relative incorporation efficiencies [(k pol/K d )incorrect/(k pol/K d )TTPcorrect] of 4.1 × 10−5 for TTP and 3.4 × 10−6 for TMeTP in case of RTWT and 1.4 × 10−5 for TTP and 2.9 × 10−8for TMeTP in case of RTM184V.

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.