Switching the proton-coupled electron transfer mechanism for non-canonical tyrosine residues in a de novo protein.

The proton-coupled electron transfer (PCET) reactions of tyrosine (Y) are instrumental to many redox reactions in nature. This study investigates how the local environment and the thermodynamic properties of Y influence its PCET characteristics. Herein, 2- and 4-mercaptophenol (MP) are placed in the well-folded α3C protein (forming 2MP-α3C and 4MP-α3C) and oxidized by external light-generated [Ru(L)3]3+ complexes. The resulting neutral radicals are long-lived (>100 s) with distinct optical and EPR spectra. Calculated spin-density distributions are similar to canonical Y˙ and display very little spin on the S-S bridge that ligates the MPs to C32 inside the protein. With 2MP-α3C and 4MP-α3C we probe how proton transfer (PT) affects the PCET rate constants and mechanisms by varying the degree of solvent exposure or the potential to form an internal hydrogen bond. Solution NMR ensemble structures confirmed our intended design by displaying a major difference in the phenol OH solvent accessible surface area (≤∼2% for 2MP and 30-40% for 4MP). Additionally, 2MP-C32 is within hydrogen bonding distance to a nearby glutamate (average O-O distance is 3.2 ± 0.5 Å), which is suggested also by quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations. Neither increased exposure of the phenol OH to solvent (buffered water), nor the internal hydrogen bond, was found to significantly affect the PCET rates. However, the lower phenol pKa values associated with the MP-α3C proteins compared to α3Y provided a sufficient change in PT driving force to alter the PCET mechanism. The PCET mechanism for 2MP-α3C and 4MP-α3C with moderately strong oxidants was predominantly step-wise PTET for pH values, but changed to concerted PCET at neutral pH values and below when a stronger oxidant was used, as found previously for α3Y. This shows how the balance of ET and PT driving forces is critical for controlling PCET mechanisms. The presented results improve our general understanding of amino-acid based PCET in enzymes.

Protease-stable DARPins as promising oral therapeutics.

Clostridioides difficile is an enteric bacterium whose exotoxins, TcdA and TcdB, inactivate small GTPases within the host cells, leading to bloody diarrhea. In prior work, our group engineered a panel of potent TcdB-neutralizing designed ankyrin repeat proteins (DARPin) as oral therapeutics against C. difficile infection. However, all these DARPins are highly susceptible to digestion by gut-resident proteases, i.e. trypsin and chymotrypsin. Close evaluation of the protein sequence revealed a large abundance of positively charged and aromatic residues in the DARPin scaffold. In this study, we significantly improved the protease stability of one of the DARPins, 1.4E, via protein engineering. Unlike 1.4E, whose anti-TcdB EC50 increased >83-fold after 1-hour incubation with trypsin (1 mg/ml) or chymotrypsin (0.5 mg/ml), the best progenies-T10-2 and T10b-exhibit similar anti-TcdB potency as their parent in PBS regardless of protease treatment. The superior protease stability of T10-2 and T10b is attributed to the removal of nearly all positively charged and aromatic residues except those directly engaged in target binding. Furthermore, T10-2 was found to retain significant toxin-neutralization ability in ex vivo cecum fluid and can be easily detected in mouse fecal samples upon oral administration. Both T10-2 and T10b enjoy a high thermo- and chemo-stability and can be expressed very efficiently in Escherichia coli (>100 mg/l in shaker flasks). We believe that, in additional to their potential as oral therapeutics against C. difficile infection, T10-2 and T10b can also serve as a new generation DARPin scaffold with superior protease stability.

Pb2+ as modulator of protein-membrane interactions.

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.

Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine 40 of tyrosine hydroxylase on the conformation of its regulatory domain.

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate constant for dissociation of inhibitory catecholamines from the active site by 3 orders of magnitude. To probe the changes in the structure of the N-terminal domain upon phosphorylation, individual phenylalanine residues at positions 14, 34, and 74 were replaced with tryptophan in a form of the protein in which the endogenous tryptophans had all been mutated to phenylalanine (W(3)F TyrH). The steady-state fluorescence anisotropy of F74W W(3)F TyrH was unaffected by phosphorylation, but the anisotropies of both F14W and F34W W(3)F TyrH increased significantly upon phosphorylation. The fluorescence of the single tryptophan residue at position 74 was less readily quenched by acrylamide than those at the other two positions; fluorescence increased the rate constant for quenching of the residues at positions 14 and 34 but did not affect that for the residue at position 74. Frequency domain analyses were consistent with phosphorylation having no effect on the amplitude of the rotational motion of the indole ring at position 74, resulting in a small increase in the rotational motion of the residue at position 14 and resulting in a larger increase in the rotational motion of the residue at position 34. These results are consistent with the local environment at position 74 being unaffected by phosphorylation, that at position 34 becoming much more flexible upon phosphorylation, and that at position 14 becoming slightly more flexible upon phosphorylation. The results support a model in which phosphorylation at Ser40 at the N-terminus of the regulatory domain causes a conformational change to a more open conformation in which the N-terminus of the protein no longer inhibits dissociation of a bound catecholamine from the active site.

Impact of hapten presentation on antibody binding at lipid membrane interfaces.

We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K(D)) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, K(D)(bulk) = 1.7 +/- 0.2 nM vs. 2.9 +/- 0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, K(D) = 3.6 +/- 1.1 nM vs. 2.0 +/- 0.2 microM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands' relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the K(D) value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4 +/- 1.1 microM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (K(D) = 21 +/- 10 nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands.

Tryptophan fluorescence reveals the presence of long-range interactions in the denatured state of ribonuclease Sa.

Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues. We made five variants of RNase Sa by adding Trp residues at locations where they are found in other members of the microbial ribonuclease family. To better understand the protein denatured state, we also studied the fluorescence properties of the following peptides: N-acetyl-Trp-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and the five pentapeptides with the same sequence as the Trp substitution sites in RNase Sa. The major conclusions are: 1), the wavelength of maximum fluorescence intensity, lambda(max), does not differ significantly for the peptides and the denatured proteins; 2), the fluorescence intensity at lambda(max), I(F), differs significantly for the five Trp containing variants of RNase Sa; 3), the I(F) differences for the denatured proteins are mirrored in the peptides, showing that the short-range effects giving rise to the I(F) differences in the peptides are also present in the proteins; 4) the I(F) values for the denatured proteins are more than 30% greater than for the peptides, showing the presence of long-range effects in the proteins; 5), fluorescence quenching of Trp by acrylamide and iodide is more than 50% greater in the peptides than in the denatured proteins, showing that long-range effects limit the accessibility of the quenchers to the Trp side chains in the proteins; and 6), these results show that nonlocal effects in the denatured states of proteins influence Trp fluorescence and accessibility significantly.

Peptide sequence and conformation strongly influence tryptophan fluorescence.

This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.

IIAGlc inhibition of glycerol kinase: a communications network tunes protein motions at the allosteric site.

Steady-state and time-resolved fluorescence anisotropy methods applied to an extrinsic fluorophore that is conjugated to non-native cysteine residues demonstrate that amino acids in an allosteric communication network within a protein subunit tune protein backbone motions at a distal site to enable allosteric binding and inhibition. The unphosphorylated form of the phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli glycerol kinase, binding more than 25 A from the kinase active site. Crystal structures that showed a ligand-dependent conformational change and large temperature factors for the IIAGlc-binding site on E. coli glycerol kinase suggest that motions of the allosteric site have an important role in the inhibition. Three E. coli glycerol kinase amino acids that are located at least 15 A from the active site and the allosteric site were shown previously to be necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol kinase from Haemophilus influenzae. These three amino acids are termed the coupling locus. The apparent allosteric site motions and the requirement for the distant coupling locus to transplant allosteric inhibition suggest that the coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. influenzae glycerol kinase were constructed with a non-native cysteine residue replacing one of the native residues in the IIAGlc-binding site. The extrinsic fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated specifically to the non-native cysteine residue. Steady-state and time-resolved fluorescence anisotropy measurements show that the motions of the fluorophore reflect backbone motions of the IIAGlc-binding site and these motions are modulated by the amino acids at the coupling locus.

Effects of ligands on the mobility of an active-site loop in tyrosine hydroxylase as monitored by fluorescence anisotropy.

Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop over the active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, and the three native tryptophan residues were mutated to phenylalanine to form an enzyme with a single tryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzyme resulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did not result in a significant change in the anisotropy in the presence or absence of the deazapterin. The K(d) value for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar results were obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease in anisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes was unaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistent with a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could be modeled as a decrease in the cone angle for the indole ring of about 12 degrees . The data are consistent with a model in which binding of a tetrahydropterin results in a change in the conformation of the surface loop required for proper formation of the amino acid binding site.

Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa.

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.