Eyal Arbely

Photocontrol of tyrosine phosphorylation in mammalian cells via genetic encoding of photocaged tyrosine.

We report the first site-specific genetic encoding of photocaged tyrosine into proteins in mammalian cells. By photocaging Tyr701 of STAT1 we demonstrate that it is possible to photocontrol tyrosine phosphorylation and signal transduction in mammalian cells.

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-Å resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNA(p53DBD)4 or DNA(AcK120DBD)4, indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.

A highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein.

Abstract The agent responsible for the recent severe acute respiratory syndrome (SARS) outbreak is a previously unidentified coronavirus. While there is a wealth of epidemiological studies, little if any molecular characterization of SARS coronavirus (SCoV) proteins has been carried out. Here we describe the molecular characterization of SCoV E protein, a critical component of the virus responsible for virion envelope morphogenesis. We conclusively show that SCoV E protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to SCoV. The hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of E proteins pivotal role in viral budding. The molecular understanding of this critical component of SCoV may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity.

Downhill versus barrier-limited folding of BBL 1: energetic and structural perturbation effects upon protonation of a histidine of unusually low pKa

A dispersion of melting temperatures at pH5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH7. To search for possible causes of anomalies in BBLs denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK(a) of His142 was close to that of free histidine at 6.47+/-0.04, while that of the more buried His166 was highly perturbed at 5.39+/-0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by approximately 1.5 kcal/mol. Changes in C(alpha) secondary shifts at pH5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1+/-0.2 kcal/mol in stability from pH7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only approximately 1% of the structurally perturbed state will be present at the biologically relevant pH7.6. Experiments at pH5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK(a) induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH5.3.

SARS coronavirus E protein in phospholipid bilayers : An X-ray study

Abstract We investigated the structure of the hydrophobic domain of the severe acute respiratory syndrome E protein in model lipid membranes by x-ray reflectivity and x-ray scattering. In particular, we used x-ray reflectivity to study the location of an iodine-labeled residue within the lipid bilayer. The label imposes spatial constraints on the protein topology. Experimental data taken as a function of protein/lipid ratio P/L and different swelling states support the hairpin conformation of severe acute respiratory syndrome E protein reported previously. Changes in the bilayer thickness and acyl-chain ordering are presented as a function of P/L, and discussed in view of different structural models.

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed (13)C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK(a) values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK(a) values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK(a), and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured (13)C(α) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.

Modeling Sample Disorder in Site-Specific Dichroism Studies of Uniaxial Systems

Site-specific infrared dichroism is an emerging method capable of proposing a model for the backbone structure of a transmembrane alpha-helix within a helical bundle. Dichroism measurements of single, isotopically enhanced vibrational modes (e.g., Amide I 13C=18O or Gly CD2 stretching modes) can yield precise orientational restraints for the monomer helix protomer that can be used as refinement constraints in model building of the entire helical bundle. Essential, however, for the interpretation of the dichroism measurements, is an accurate modeling of the sample disorder. In this study we derive an enhanced and more realistic modeling of the sample disorder based on a Gaussian distribution of the chromophore around a particular angle. The enhanced utility of the Gaussian model is exemplified by the comparative data analysis based on the aforementioned model to previously employed models.

Viral ion channel proteins in model membranes: a comparative study by X-ray reflectivity

We have investigated the effect of the transmembrane domain of three viral ion channel proteins on the lipid bilayer structure by X-ray reflectivity and scattering from oriented planar bilayers. The proteins show a similar effect on the lipid bilayer structural parameters: an increase in the lipid bilayer hydrophobic core, a decrease in the amplitude of the vertical density profile and a systematic change in the ordering of the acyl chains as a function of protein-to-lipid ratio. These results are discussed in a comparative view.

SARS E protein in phospholipid bilayers: an anomalous X-ray reflectivity study

Abstract We report on an anomalous X-ray reflectivity study to locate a labelled residue of a membrane protein with respect to the lipid bilayer. From such experiments, important constraints on the protein or peptide conformation can be derived. Specifically, our aim is to localize an iodine-labelled phenylalanine in the SARS E protein, incorporated in DMPC phospholipid bilayers, which are deposited in the form of thick multilamellar stacks on silicon surfaces. Here, we discuss the experimental aspects and the difficulties associated with the Fourier synthesis analysis that gives the electron density profile of the membranes.

Site-Specific Dichroism Analysis Utilizing Transmission FTIR

Infrared spectroscopy has long been used to examine the average secondary structure and orientation of membrane proteins. With the recent utilization of site-specific isotope labeling (e.g., peptidic 1-(13)C = (18)O) it is now possible to examine localized properties, rather than global averages. The technique of site-specific infrared dichroism (SSID) capitalized on this fact, and derives site-specific orientational restraints for the labeled amino acids. These restraints can then be used to solve the backbone structure of simple alpha-helical bundles, emphasizing the capabilities of this approach. So far SSID has been carried out in attenuated total internal reflection optical mode, with all of the respective caveats of attenuated total internal reflection. In this report we extend SSID through the use of transmission infrared spectroscopy tilt series. We develop the corresponding theory and demonstrate that accurate site-specific orientational restraints can be derived from a simple transmission experiment.