Alternative type I and I' turn conformations in the beta8/beta9 beta-hairpin of human acidic fibroblast growth factor

Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects.     

Crystal structure of the predicted phospholipase LYPLAL1 reveals unexpected functional plasticity despite close relationship to acyl protein thioesterases

Sequence homology indicates the existence of three human cytosolic acyl protein thioesterases, including APT1 that is known to depalmitoylate H- and N-Ras. One of them is the lysophospholipase-like 1 (LYPLAL1) protein that on the one hand is predicted to be closely related to APT1 but on the other hand might also function as a potential triacylglycerol lipase involved in obesity. However, its role remained unclear. The 1.7 Å crystal structure of LYPLAL1 reveals a fold very similar to APT1, as expected, but features a shape of the active site that precludes binding of long-chain substrates. Biochemical data demonstrate that LYPLAL1 exhibits neither phospholipase nor triacylglycerol lipase activity, but rather accepts short-chain substrates. Furthermore, extensive screening efforts using chemical array technique revealed a first small molecule inhibitor of LYPLAL1.     

Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+

The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a beta beta alpha-metal topology. Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni2+-bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 A and 2.0 A, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ at concentrations of <25 mM, indicating that zinc is the physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P-O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.     

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.     

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.     

IKKalpha, a critical regulator of epidermal differentiation and a suppressor of skin cancer

IκB kinase α (IKKα), one of the two catalytic subunits of the IKK complex involved in nuclear factor κB (NF-κB) activation, also functions as a molecular switch that controls epidermal differentiation. This unexpected function requires IKKα nuclear translocation but does not depend on its kinase activity, and is independent of NF-κB signalling. Ikkα^–/– mice present with a hyperproliferative and undifferentiated epidermis characterized by complete absence of a granular layer and stratum corneum. Ikkα-deficient keratinocytes do not express terminal differentiation markers and continue to proliferate even when subjected to differentiation-inducing stimuli. This antiproliferative function of IKKα is also important for the suppression of squamous cell carcinogenesis. The exact mechanisms by which nuclear IKKα controls keratinocyte proliferation and differentiation remained mysterious for some time. Recent studies, however, have revealed that IKKα is a major cofactor in a TGFβ–Smad2/3 signalling pathway that is Smad4 independent. This pathway controls cell cycle withdrawal during keratinocyte terminal differentiation. Although these are not the only functions of nuclear IKKα, this multifunctional protein is a key regulator of keratinocyte and epidermal differentiation and a critical suppressor of skin cancer.     

Long-range side-chain-main-chain interactions play crucial roles in stabilizing the (betaalpha)8 barrel motif of the alpha subunit of tryptophan synthase

The role of hither-to-fore unrecognized long-range hydrogen bonds between main-chain amide hydrogens and polar side chains on the stability of a well-studied (betaalpha)8, TIM barrel protein, the alpha subunit of tryptophan synthase (alphaTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a beta-strand with the C terminus of the succeeding antiparallel alpha-helix, and the A103-D130 hydrogen bond links the N terminus of an alpha-helix with the C terminus of the succeeding antiparallel beta-strand, forming clamps for the respective betaalpha or alphabeta hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far-UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main-chain-side-chain hydrogen bond, G26-S33, and two electrostatic side-chain-side-chain hydrogen bonds, D38-H92 and D112-H146, all in the same N-terminal folding unit of alphaTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.     

Factors involved in the stability of isolated beta-sheets: Turn sequence, beta-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.     

Simulation of pH-dependent edge strand rearrangement in human beta-2 microglobulin

Amyloid fibrils formed from unrelated proteins often share morphological similarities, suggesting common biophysical mechanisms for amyloidogenesis. Biochemical studies of human beta-2 microglobulin (beta2M) have shown that its transition from a water-soluble protein to insoluble aggregates can be triggered by low pH. Additionally, biophysical measurements of beta2M using NMR have identified residues of the protein that participate in the formation of amyloid fibrils. The crystal structure of monomeric human beta2M determined at pH 5.7 shows that one of its edge beta-strands (strand D) adopts a conformation that differs from other structures of the same protein obtained at higher pH. This alternate beta-strand arrangement lacks a beta-bulge, which may facilitate protein aggregation through intermolecular beta-sheet association. To explore whether the pH change may yield the observed conformational difference, molecular dynamics simulations of beta2M were performed. The effects of pH were modeled by specifying the protonation states of Asp, Glu, and His, as well as the C terminus of the main chain. The bulged conformation of strand D is preferred at medium pH (pH 5-7), whereas at low pH (pH < 4) the straight conformation is observed. Therefore, low pH may stabilize the straight conformation of edge strand D and thus increase the amyloidogenicity of beta2M.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Conserved F84 and P86 residues in alphaB-crystallin are essential to effectively prevent the aggregation of substrate proteins

Previously, we have shown that residues 73-92 (sequence DRFSVNLDVKHFSPEELKVK) in alphaB-crystallin are involved in preventing the formation of light scattering aggregates by substrate proteins. In this study, we made single substitutions of three conserved amino acid residues (H83 --> A, F84 --> G, and P86 --> A) and a nonconserved amino acid residue (K90 --> C) in the functional region of alphaB-crystallin and evaluated their role in anti-aggregation activity. Mutation of conserved residues led to changes in intrinsic tryptophan intensity, bis-ANS binding, and in the secondary and tertiary structures. The H83A mutation led to a twofold increase in molar mass, while the other mutants did not produce significant changes in the molar mass when compared to that of wild-type protein. The chaperone-like activity of the H83A mutant was enhanced by 15%-20%, and the chaperone-like activity of F84G and P86A mutants was reduced by 50%-65% when compared to the chaperone-like activity of wild-type alphaB-crystallin. The substitution of the nonconserved residue (K90 --> C) did not induce an appreciable change in the structure and function of the mutant protein. Fluorescence resonance energy transfer (FRET) assay demonstrated that destabilized ADH interacted near the K90 region in alphaB-crystallin. The data show that F84 and P86 residues are essential for alphaB-crystallin to effectively prevent the aggregation of substrate proteins. This study further supports the involvement of the residues in the 73-92 region of alphaB-crystallin in substrate protein binding and chaperone-like action.     

Solution structure of Pi4, a short four-disulfide-bridged scorpion toxin specific of potassium channels

Pi4 is a short toxin found at very low abundance in the venom of Pandinus imperator scorpions. It is a potent blocker of K(+) channels. Like the other members of the alpha-KTX6 subfamily to which it belongs, it is cross-linked by four disulfide bonds. The synthetic analog (sPi4) and the natural toxin (nPi4) have been obtained by solid-phase synthesis or from scorpion venom, respectively. Analysis of two-dimensional (1)H NMR spectra of nPi4 and sPi4 indicates that both peptides have the same structure. Moreover, electrophysiological recordings of the blocking of Shaker B K(+) channels by sPi4 (K(D) = 8.5 nM) indicate that sPi4 has the same blocking activity of nPi4 (K(D) = 8.0 nM), previously described. The disulfide bonds have been independently determined by NMR and structure calculations, and by Edman-degradation/mass-spectrometry identification of peptides obtained by proteolysis of nPi4. Both approaches indicate that the pairing of the half-cystines is (6)C-(27)C, (12)C-(32)C, (16)C-(34)C, and (22)C-(37)C. The structure of the toxin has been determined by using 705 constraints derived from NMR data on sPi4. The structure, which is well defined, shows the characteristic alpha/beta scaffold of scorpion toxins. It is compared to the structure of the other alpha-KTX6 subfamily members and, in particular, to the structure of maurotoxin, which shows a different pattern of disulfide bridges despite its high degree of sequence identity (76%) with Pi4. The structure of Pi4 and the high amounts of synthetic peptide available, will enable the detailed analysis of the interaction of Pi4 with K(+) channels.     

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.     

Identification of a region in G protein γ subunits conserved across species but hypervariable among subunit isoforms

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.     

Buried hydrophobic side-chains essential for the folding of the parallel beta-helix domains of the P22 tailspike

The processive beta-strands and turns of a polypeptide parallel beta-helix represent one of the topologically simplest beta-sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel beta-helix followed by an interdigitated section of triple-stranded beta-helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two beta-helix domains and extend into the core of the contiguous triple beta-prism domain. To test whether these side-chain stacks represent essential residues for driving the chain into the correct fold, each of three stacked phenylalanine residues within the buried core were substituted with less bulky amino acids. The mutant chains with alanine in place of phenylalanine were defective in intracellular folding. The chains accumulated exclusively in the aggregated inclusion body state regardless of temperature of folding. These severe folding defects indicate that the stacked phenylalanine residues are essential for correct parallel beta-helix folding. Replacement of the same phenylalanine residues with valine or leucine also impaired folding in vivo, but with less severity. Mutants were also constructed in a second buried stack that extends into the intertwined triple-stranded beta-helix and contiguous beta-prism regions of the protein. These mutants exhibited severe defects in later stages of chain folding or assembly, accumulating as misfolded but soluble multimeric species. The results indicate that the formation of the buried hydrophobic stacks is critical for the correct folding of the parallel beta-helix, triple-stranded beta-helix, and beta-prism domains in the tailspike protein.     

Structural basis for the topological specificity function of MinE

Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel alphabeta sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.     

Division site placement in E.coli: mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains

The MinE protein functions as a topological specificity factor in determining the site of septal placement in Escherichia coli. MinE assembles into a membrane-associated ring structure near midcell and directs the localization of MinD and MinC into a membrane- associated polar zone that undergoes a characteristic pole-to-pole oscillation cycle. Single (green fluorescent protein) and double label (yellow fluorescent protein/cyan fluorescent protein) fluorescence labeling experiments showed that mutational alteration of a site on the alpha-face of MinE led to a failure to assemble the MinE ring, associated with loss of the ability to support a normal pattern of division site placement. The absence of the MinE ring did not prevent the assembly and disassembly of the MinD polar zone. Mutant cells lacking the MinE ring were characterized by the growth of MinD polar zones past their normal arrest point near midcell. The results suggested that the MinE ring acts as a stop-growth mechanism to prevent the MinCD polar zone from extending beyond the midcell division site.     

Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the zinc-assistant hydrolysis of N-acyl-D-amino acids. We report here the cloning, expression, and structural-based mutation of the D-aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue D-aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel alpha/beta-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in D-aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization.     

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells: IMPLICATIONS FOR AGGREGATION AND CELLULAR PROPERTIES OF α-SYNUCLEIN

N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N(α)-acetylation on the biophysical and biological properties of α-syn, we produced N(α)-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909-3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and N(α)-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N(α)-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N(α)-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating N(α)-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N(α)-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells.