Residue-Specific Incorporation of Noncanonical Amino Acids for Protein Engineering.

The incorporation of noncanonical amino acids has given protein chemists access to an expanded repertoire of amino acids. This methodology has significantly broadened the scope of protein engineering allowing introduction of amino acids with non-native functionalities, such as bioorthogonal reactive handles (azides and alkynes) and hydrophobic fluorinated side chains. Here, we describe the efficient residue-specific replacement of methionine by azidonorleucine in an engineered green fluorescent protein using a bacterial expression system to introduce a single reactive site for the strain-promoted azide-alkyne cycloaddition.

A Versatile Approach for Site-Specific Lysine Acylation in Proteins.

Using amber suppression in coordination with a mutant pyrrolysyl-tRNA synthetase-tRNAPyl pair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site-specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su-H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post-translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.

Semisynthetic UbH2A reveals different activities of deubiquitinases and inhibitory effects of H2A K119 ubiquitination on H3K36 methylation in mononucleosomes.

Using a genetically incorporated azidonorleucine for ubiquitin installation, we prepared multi-milligram quantities of H2AK119ub (ubH2A). With a native isopeptide linkage, the synthetic ubH2A was used to study the activity of deubiquitinases and crosstalk between H2A ubiquitination and H3K36 methylation in the context of chemically defined mononucleosomes.

Making connections between ultrafast protein folding kinetics and molecular dynamics simulations.

Determining the rate of forming the truly folded conformation of ultrafast folding proteins is an important issue for both experiments and simulations. The double-norleucine mutant of the 35-residue villin subdomain is the focus of recent computer simulations with atomistic molecular dynamics because it is currently the fastest folding protein. The folding kinetics of this protein have been measured in laser temperature-jump experiments using tryptophan fluorescence as a probe of overall folding. The conclusion from the simulations, however, is that the rate determined by fluorescence is significantly larger than the rate of overall folding. We have therefore employed an independent experimental method to determine the folding rate. The decay of the tryptophan triplet-state in photoselection experiments was used to monitor the change in the unfolded population for a sequence of the villin subdomain with one amino acid difference from that of the laser temperature-jump experiments, but with almost identical equilibrium properties. Folding times obtained in a two-state analysis of the results from the two methods at denaturant concentrations varying from 1.5-6.0 M guanidinium chloride are in excellent agreement, with an average difference of only 20%. Polynomial extrapolation of all the data to zero denaturant yields a folding time of 220 (+100,-70) ns at 283 K, suggesting that under these conditions the barrier between folded and unfolded states has effectively disappeared--the so-called "downhill scenario."

Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

Following the studies of urea denaturation of ß-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of ß-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues.

We replaced the single N-terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non-canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non-canonical amino acid incorporation protocol involves a two-step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of approximately 12% could be elevated to 40% by increasing the non-canonical amino acid concentration in the medium by 10-fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture.

Substitution of methionine 35 inhibits apoptotic effects of Abeta(31-35) and Abeta(25-35) fragments of amyloid-beta protein in PC12 cells.

BACKGROUND: Amyloid-beta peptide (AbetaP), the central constituent of senile plaques in Alzheimer's disease (AD) brain, has been shown to be toxic to neuronal cells and that this toxicity is responsible for the progressive cognitive decline associated with this neurodegenerative disease. The precise mechanism of AbetaP action remains to be determined; however, it has been reported that the methionine residue at position 35 plays a pivotal role in the toxicity of the peptide. With this in mind, the present study examines the effect of mutating the methionine to norleucine in the fragments (31-35) and (25-35) of AbetaP, which have methionine at the C-terminal, in order to investigate the influence of this residue on Abeta-mediated toxic effects on PC12 cells. MATERIAL/METHODS: The toxic and apoptotic effects (release of Cyt c, caspase activation, and DNA fragmentation) exerted by the Abeta(31-35) and Abeta(25-35) wild-type peptides and Abeta(31-35)Met-->Nle, Abeta(25-35)Met-->Nle peptides where methionine was substituted with norleucine were investigated on PC12 cells. RESULTS: The results obtained shown that both peptides induce neurotoxicity in PC12 cells via an apoptotic cell death pathway, including cytochrome c release, caspase activation, and DNA fragmentation. Furthermore, this study reveals that these events were completely abrogated in cells exposed to Abeta peptides in which methionine 35 was substituted by a norleucine residue. CONCLUSIONS: On the basis of the results obtained in this study, an additional hypothesis involving the amyloid-beta peptide and the role of Met-35 has been proposed to clarify the mechanisms responsible of neurodegeneration in Alzheimer's disease.

High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein.

The 35-residue subdomain of the villin headpiece (HP35) is a small ultrafast folding protein that is being intensely studied by experiments, theory, and simulations. We have solved the x-ray structures of HP35 and its fastest folding mutant [K24 norleucine (nL)] to atomic resolution and compared their experimentally measured folding kinetics by using laser temperature jump. The structures, which are in different space groups, are almost identical to each other but differ significantly from previously solved NMR structures. Hence, the differences between the x-ray and NMR structures are probably not caused by lattice contacts or crystal/solution differences, but reflect the higher accuracy of the x-ray structures. The x-ray structures reveal important details of packing of the hydrophobic core and some additional features, such as cross-helical H bonds. Comparison of the x-ray structures indicates that the nL substitution produces only local perturbations. Consequently, the finding that the small stabilization by the mutation is completely reflected in an increased folding rate suggests that this region of the protein is as structured in the transition state as in the folded structure. It is therefore a target for engineering to increase the folding rate of the subdomain from approximately 0.5 micros(-1) for the nL mutant to the estimated theoretical speed limit of approximately 3 micros(-1).

Determinants of high-affinity binding and receptor activation in the N-terminus of CCL-19 (MIP-3 beta).

CC chemokine receptor 7 (CCR-7) is expressed on mature dendritic cells and T-cells. Its ligands, CCL-19 (MIP-3beta) and CCL-21 (SLC), play an important role in the migration of these cells to secondary lymphoid organs where they are predominantly expressed. For most chemokines, the N-terminal domain preceding the first two conserved cysteines is involved in stabilizing the active conformation of its cognate receptors. We have chemically synthesized N-terminal analogues of CCL-19 with the aid of a native chemical ligation method to investigate structure function requirements of this ligand domain by performing ligand binding, GTP-gammaS binding, and chemotaxis assays. Successive truncations of the N-terminus of CCL-19 reduced the affinity of the receptor for the ligand in a size-dependent manner. Furthermore, Ala substitutions of Asn(3), Asp(4), and Asp(7) show that the side chains of these residues are important for high-affinity binding of CCL-19 to CCR-7. The effects observed were mirrored in both GTP-gammaS binding and chemotaxis assays, highlighting the functional importance of this ligand domain. We also describe two partial agonists of CCR-7 ([Nle(72)]CCL-19(6-77) and Ac-[Nle(72)]CCL-19(7-77)), and identify the first analogue of CCL-19 (Ac-[Nle(72)]CCL-19(8-77)) that acts as a functional antagonist in vitro (K(B) approximately 350 nM for GTP-gammaS binding assays). As mutations of both Glu(6) and Asp(7) to Ala did not dissociate effects on ligand binding from receptor activation, it is likely that the backbone of these two residues is crucial for agonist activity.

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges.

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.

Rational modification of protein stability by the mutation of charged surface residues.

Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-residue helical protein, the peripheral subunit-binding domain. The results of these calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of these calculations. Arg8 is located on the surface of the protein in a region of positive electrostatic potential. The calculations suggest that Arg8 makes a significant, unfavorable electrostatic contribution to the overall stability. The experiments described in this paper represent the first direct experimental test of the theoretical methods, taking advantage of solid-phase peptide synthesis to incorporate approximately isosteric amino acid substitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with alpha-amino adipic acid (Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effects of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the validity of the calculations. These replacements led to stability increases at 52.6 degrees C, the T(m) of the wild-type, of 0.86 and 1.08 kcal mol(-)(1), respectively. The stability of Arg8Nle is particularly interesting as a rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stability of the protein.

The invariant ARG91 is required for the rupture of liposomes by cytochrome C.

Lipid binding properties of cytochrome c (cyt c) were investigated by using semisynthetic mutant protein having amino acid substitution on the evolutionary invariant residue Arg91. We demonstrate here that the membrane binding properties of cyt c are dramatically altered by substituting norleucine for the invariant Arg91. More specifically, while the binding of this mutant to liposomes per se is indistinguishable from the wild type protein, it completely lacks the ability of the native cyt c to rupture liposome bilayers.