Design and characterization of stimuli-responsive FLAG-tag analogues and the illumination-induced modulation of their interaction with antibody 4E11

An azobenzene group containing beta-amino acid N-Fmoc-4-aminomethyl phenylazobenzoic acid was synthesized and with the exception of the C-terminal amino acid residue was substituted by solid-phase peptide synthesis into all positions of the FLAG sequence (DYKDDDDK), an octapeptide capable of specific interaction with the monoclonal antibody 4E11. The trans state of the beta-amino acid was thermodynamically more stable than the cis state. However, the molecule could be switched into the cis conformation by illumination at 340 nm. Peptides containing the artificial amino acid also became photoresponsive. In the absence of light, the spontaneous back-isomerization into the trans conformation of the photoresponsive was extremely slow (>8 h no significant increase in trans content). When illuminated with visible light (440 nm), the back-isomerization from the cis to the trans state was accelerated and occurred with a half-life of approximately 10 min. The cis form of the photopeptides was more hydrophilic than the trans form, as evidenced by differences in the retention time of the two isomeric forms in reversed-phase chromatography. Photopeptides that contained the intact sequences responsible for binding of the FLAG tag to the antibody, namely, the DYK motive at the N-terminus, showed binding to the antibody in both a dot blot immunoassay and in Biacore binding studies, albeit with lower affinity than the unmodified FLAG sequence. Peptides with a substitution in positions 4-6 showed differences in binding strength between the trans and the cis form in the Biacore studies, no such difference could be observed for the peptide with a substitution in position 7.     

Photomodulation of conformational states. IV. Integrin-binding RGD-peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

In previous studies we have investigated octapeptides backbone-cyclized by (4-amino)phenyl azobenzoic acid (APB) or (4-aminomethyl)phenylazobenzoic acid (AMPB) and containing the active-site sequence Cys-Ala-Thr-Cys-Asp from the thioredoxin reductase. The conformational and redox properties of these peptides were strongly dependent on the isomeric state of the azobenzene chromophore. Using the same approach we were successful in constructing photoresponsive ligands for alphavbeta3 integrin containing the Arg-Gly-Asp (RGD) sequence as binding motif. For achieving maximal conformational restriction of the peptide a reduced ring size compared to our previous azobenzene peptides was employed in the cyclic peptide c[Asp-D-Phe-Val-AMPB-Lys-Ala-Arg-Gly-]. Conformational properties of the trans and cis isomers of this peptide in solution were investigated by CD and NMR and were found to differ markedly from the thioredoxin derived azobenzene peptides. In a second peptide, c[Asp-D-Phe-Val-Lys-AMPB-Ala-Arg-Gly-], shifting the position of the chromophore lead to a marked decrease in affinity. With the availability of the x-ray structure of a cyclic RGD-pentapeptide bound to alphavbeta3 integrin (PDB entry 1L5G) modeling of possible bound conformations for trans and cis isomers of both azobenzene peptides was possible. Notably, both peptides in either isomeric form share the same overall conformation in the bound state according to our molecular dynamics simulations.     

Photomodulation of conformational states. III. Water-soluble bis-cysteinyl-peptides with (4-aminomethyl) phenylazobenzoic acid as backbone constituent

In previous studies we have shown that light-induced cis/trans isomerization of the azobenzene moiety in cyclo-[Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-AMPB] [AMPB: (4-aminomethyl)phenylazobenzoic acid] leads both in the monocyclic and in the oxidized bicyclic form to markedly differentiated conformational states in DMSO, a fact that lends itself for photomodulation of the redox potential of such bis-cysteinyl-peptides. For this purpose water-soluble systems are required, and this was achieved by replacing three residues outside the Cys-Ala-Thr-Cys active-site motif of thioredoxin reductase with lysines. The resulting cyclo-[Lys-Cys-Ala-Thr-Cys-Asp-Lys-Lys-AMPB] fully retains its photoresponsive properties in water as well assessed by uv and CD measurements. Paralleling results of the previously investigated azobenzene-containing cyclic peptides, the trans --> cis isomerization of the water-soluble monocyclic and oxidized bicyclic peptide is accompanied by a marked transition from a well-defined conformation to an ensemble of possible conformations. However, the conformational preferences are very dissimilar from those of the DMSO-soluble peptides. In fact, hydrogen bonds as well as secondary structure elements were found that change in the mono- and bicyclic peptide upon irradiation. The photo switch between different turn types and hydrogen bonding networks offers the structural rational for the significantly differentiated redox potentials, but also the possibility of monitoring by femtosecond uv-vis and ir spectroscopy fast and ultra fast backbone rearrangement processes following the electronic trans --> cis isomerization.     

Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides

The synthesis of an azobenzene amino acid (aa) for use as a photo-inducible conformational switch in polypeptides is described. The compound can be easily incorporated into an aa sequence by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl methods. A reversible conformational change of the peptide backbone is induced by switching between the cis and trans configurations of the azobenzene moiety by irradiation with light of suitable wavelength. Thermal cis --> trans isomerization of this azobenzene aa is slow, enabling detailed structural investigations of the modified peptides, e.g., using NMR techniques. The total time for the synthesis of the photoswitch is typically 4 d, with an overall yield of 40-50%.     

Photoreversible antigen-antibody reactions

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.     

Structural mechanism governing cis and trans isomeric states and an intramolecular switch for cis/trans isomerization of a non-proline peptide bond observed in crystal structures of scorpion toxins

Non-proline cis peptide bonds have been observed in numerous protein crystal structures even though the energetic barrier to this conformation is significant and no non-prolyl-cis/trans-isomerase has been identified to date. While some external factors, such as metal binding or co-factor interaction, have been identified that appear to induce cis/trans isomerization of non-proline peptide bonds, the intrinsic structural basis for their existence and the mechanism governing cis/trans isomerization in proteins remains poorly understood. Here, we report the crystal structure of a newly isolated neurotoxin, the scorpion alpha-like toxin Buthus martensii Karsch (BmK) M7, at 1.4A resolution. BmK M7 crystallizes as a dimer in which the identical non-proline peptide bond between residues 9 and 10 exists either in the cis conformation or as a mixture of cis and trans conformations in either monomer. We also determined the crystal structures of several mutants of BmK M1, a representative scorpion alpha-like toxin that contains an identical non-proline cis peptide bond as that observed in BmK M7, in which residues within or neighboring the cis peptide bond were altered. Substitution of an aspartic acid residue for lysine at residue 8 in the BmK M1 (K8D) mutant converted the cis form of the non-proline peptide bond 9-10 into the trans form, revealing an intramolecular switch for cis-to-trans isomerization. Cis/trans interconversion of the switch residue at position 8 appears to be sequence-dependent as the peptide bond between residues 9 and 10 retains its wild-type cis conformation in the BmK M1 (K8Q) mutant structure. The structural interconversion of the isomeric states of the BmK M1 non-proline cis peptide bond may relate to the conversion of the scorpion alpha-toxins subgroups.     

Ultrafast conformational dynamics in cyclic azobenzene peptides of increased flexibility

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.     

Photomodulation of conformational states. I. Mono- and bicyclic peptides with (4-amino)phenylazobenzoic acid as backbone constituent

The thioredoxin reductase active-site fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141], which is known for its high tendency to assume an almost identical conformation as in the intact enzyme, was backbone cyclized with the photoresponsive (4-amino)phenylazobenzoic acid (APB) to produce a monocyclic and disulfide-bridged bicyclic APB-peptide. Light-induced reversible cis/trans isomerization occurs at identical extents in both the linear and the two cyclic forms. Nuclear magnetic resonance conformational analysis clearly revealed that in the bicyclic APB-peptide both as a trans- and cis-azo-isomer the constraints imparted by the bicyclic structure do not allow the molecule to relax into a defined low energy conformation, thus making the molecule a frustrated system that flip-flops between multiple conformational states. Conversely, the monocyclic APB peptide folds into a well-defined lowest energy structure as a trans-azo-isomer, which upon photoisomerization to the cis-azo configuration relaxes into a less restricted conformational space. First femtosecond spectroscopic analysis of the dynamics of the photoreaction confirm a fast first phase on the femtosecond time scale related to the cis/trans isomerization of the azobenzene moiety followed by a slower phase in the picosecond time scale that involves an adjustment of the peptide backbone. Due to the well- defined photoresponsive two-state transition of this monocyclic peptide molecule, it represents a model system well suited for studying the ultrafast dynamics of conformational transitions by time-resolved spectroscopy.     

Photomodulation of conformational states. II. Mono- and bicyclic peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to an ensemble of possible conformations.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Conformation of the 18-23 loop region of bovine prothrombin in the absence and presence of a model Ca2+ ion. An energy minimization study

The conformation of the cyclic peptide Ac-Cys-Leu-Gla-Gla-Pro-Cys-NHMe, representing the 18-23 disulfide loop of bovine prothrombin, was studied by energy minimization with the ECEPP (Empirical Conformational Energy Program for Peptides) algorithm. Parameters for charge and geometry for the gamma-carboxyglutamic acid (Gla) residue were obtained for inclusion in the ECEPP data set. Construction of the 18-23 cyclic peptide, for which no crystal structure is available, was carried out by using a scheme that took advantage of the constraints imposed by the requirement of disulfide ring closure and utilized known low-energy structures of single residues and dipeptides. Both cis and trans isomers about the Gla 21-Pro 22 peptide bond were considered. The lowest-energy conformation found for the isolated 18-23 cyclic peptide with arbitrary reduction of the charge on the Gla residues (to simulate hydration roughly) is a trans form, differing in energy by 11 kcal-mol-1 from the lowest-energy cis form. However, when the energy calculation includes one model Ca2+ ion, X2+, introduced at a fixed distance of 2.3 A from a single oxygen atom of either of the side-chain carboxyl groups of Gla with the C delta-O-X2+ bond angle fixed at one of three values, the lowest-energy cis conformation is about 1 kcal-mol-1 lower in energy than the lowest-energy trans conformation; i.e. the two structures have similar energies. In these structures, four oxygen atoms, two from each Gla side-chain, approach the model Ca2+ ion closely, in a manner similar to that seen in crystals of calcium alpha-ethylmalonate (Zell, A., Einspahr, H. & Bugg, C.E. (1985) Biochemistry 24, 533-537). It appears that the binding of Ca2+ to the 18-23 cyclic peptide may alter the equilibrium between cis and trans structures such that the fraction of cis isomers is greater in the presence of Ca2+.     

Stabilization of a strained protein loop conformation through protein engineering.

Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-117 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the beta-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of K116E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "beta-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.     

Conservation of cis prolyl bonds in proteins during evolution

In proteins and peptides, the vast majority of peptide bonds occurs in trans conformation, but a considerable fraction (about 5%) of X-Pro bonds adopts the cis conformation. Here we study the conservation of cis prolyl residues in evolutionary related proteins. We find that overall, in contrast to local, protein sequence similarity is a clear indicator for the conformation of prolyl residues. We observe that cis prolyl residues are more often conserved than trans prolyl residues, and both are more conserved than the surrounding amino acids, which show the same extent of conservation as the whole protein. The pattern of amino acid exchanges differs between cis and trans prolyl residues. Also, the cis prolyl bond is maintained in proteins with sequence identity as low as 20%. This finding emphasizes the importance of cis peptide bonds in protein structure and function.     

Single proline residues can dictate the oxidative folding pathways of cysteine-rich peptides

The cysteine-rich N and C-terminal domains of minicollagen-1 from Hydra nematocysts fold with excesses of oxidized/reduced glutathione (10:1) into globular structures with distinct cystine frameworks despite their identical cysteine sequence pattern. An additional main difference is the cis conformation of a conserved proline residue in the N-terminal and the trans conformation of this residue in the C-terminal domain. Comparative analysis of the oxidative folding revealed for the C-terminal domain a fast and highly cooperative formation of a single disulfide isomer. Conversely, oxidation of the N-terminal domain proceeds mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. The rate of conversion of the bead-like isomer into the globular end-product is largely dominated by the trans-to-cis isomerization of the critical proline residue as well assessed by its replacement with (4R)- and (4S)-fluoroproline known to exhibit distinct propensities for the trans and cis conformation, respectively. Independently, whether the trans or cis conformation is favored by these substitutions, both analogues retain sufficient sequence-encoded information to fold almost quantitatively into the identical cystine framework and thus spatial structure of the parent peptide with the critical proline residue as cis isomer, but at rates significantly lower for the (4R) than for the (4S)-fluoroproline analogue. Correspondingly, other sequence-encoded structural elements have to act as a driving force for these unidirectional folding pathways despite the rather simple sequence composition consisting only of aliphatic residues, some proline and only one aromatic residue (tyrosine) in the core parts of the C and N-terminal domains. The two cysteine-rich domains of minicollagen-1 may well represent ideal targets for ab initio structure calculations in order to learn more about the elementary information encoded in such primordial molecules.     

Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.     

Photocontrol of protein folding: the interaction of photosensitive surfactants with bovine serum albumin

The photoresponsive interaction of light-sensitive azobenzene surfactants with bovine serum albumin (BSA) at neutral pH has been investigated as a means to control protein folding with light irradiation. The cationic azobenzene surfactant undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, with the visible-light (trans) form of the surfactant being more hydrophobic than the UV-light (cis) form. As a consequence, the trans form exhibits enhanced interaction with the protein compared to the cis form of the surfactant, allowing photoreversible control of the protein folding/unfolding phenomena. Small-angle neutron-scattering (SANS) measurements are used to provide detailed information on the protein conformation in solution. A fitting of the protein shape to a low-resolution triaxial ellipsoid model indicates that three discrete forms of the protein exist in solution depending on the surfactant concentration, with lengths of approximately 90, 150, and 250 A, respectively, consistent with additional dynamic light-scattering measurements. In addition, shape-reconstruction methods are applied to the SANS data to obtain relatively high-resolution conformation information. The results confirm that BSA adopts a heart-shaped structure in solution at low surfactant concentration, similar to the well-known X-ray crystallographic structure. At intermediate surfactant concentrations, protein elongation results as a consequence of the C-terminal portion separating from the rest of the molecule. Further increases in the surfactant concentration eventually lead to a highly elongated protein that nonetheless still exhibits some degree of folding that is consistent with the literature observations of a relatively high helical content in denatured BSA. The results clearly demonstrate that the visible-light form of the surfactant causes a greater degree of protein unfolding than the UV-light form, providing a means to control protein folding with light that, within the resolution of SANS, appears to be completely reversible.     

磷酸化对多肽中Xaa-Pro片段肽脯酰胺键顺反异构的影响研究

多肽和蛋白质中Xaa-Pro片段肽脯酰胺键顺反异构对其构象与功能有重要影响.设计合成了一系列模型多肽及其磷酸化多肽,并采用核磁共振实验和分子动力学模拟的方法,研究了所合成多肽中肽脯酰胺键的顺反异构化.结果表明,对脯氨酸之前的Xaa残基进行侧链O-磷酸化会极大地影响该顺反异构化过程,进而调节肽链构象.此外,磷酸化使得多肽顺式构象比例增加,且当磷酸基团不带负电荷时顺式构象所占比例最大.同时,分子动力学模拟所得结果与核磁共振实验相一致,包括最稳定构象和顺反构象统计分布.磷酸基团所带电荷及其空间位阻可能是影响这类磷酸化多肽构象变化的主要因素.     

Local and nonlocal environments around Cis peptides

Although the vast majority of peptide bonds in folded proteins are found in the trans conformation, a small percentage are found in the less energetically favorable cis conformation. Though the mechanism of cis peptide bond formation remains unknown, the role of local aromatics has been emphasized in the literature. This paper presents results from a comprehensive statistical analysis of both the local and nonlocal (i.e., tertiary) environment around cis peptides. In addition to an increased frequency of aromatic residues in the local environment around cis peptides, a number of nonlocal differences in protein secondary and tertiary structure between cis and trans peptides are found: (i) coil regions containing cis peptides are almost twice as long as those without cis peptides and include more Tyr and Pro residues; (ii) cis peptides occur with high frequencies in coil regions near large beta-structures; (iii) there is a nonlocal enrichment of Cys, His, Tyr, and Ser in the tertiary environment surrounding cis peptides when compared to trans peptides; and (iv) on average, cis peptides make fewer medium-range and more long-range contacts than trans peptides do. On the basis of these observations, it is concluded that nonlocal factors play a significant role in cis peptide formation, which has not been fully appreciated previously. An autocatalytic model for cis peptide formation is discussed as are consequences for protein folding.     

Cis phosphorylated tau as the earliest detectable pathogenic conformation in Alzheimer disease,offering novel diagnostic and therapeutic strategies

After protein phosphorylation on certain serine or threonine residues preceding a proline (pSer/Thr-Pro), the function of certain phosphorylated protein is further regulated by cis-trans conformational change. Due to the lack of any tool to detect such two conformations in cells, however, it is not even known whether any cis or trans conformation exists in vivo, not to mention their conformation-specific functions or regulation. We developed a novel peptide chemistry technology to generate the first pair of antibodies that can distinguish cis from trans pThr231-Pro tau. Cis, but not trans, pThr231-tau appears early in mild cognitive impairment (MCI) neurons and further accumulates in only degenerating neurons as Alzheimer disease (AD) progresses, localizing to dystrophic neurites, which are known to correlate well with memory loss. Unlike trans p-tau, the cis cannot promote microtubule assembly, and is more resistant to dephosphorylation and degradation and more prone to aggregation. Pin1 accelerates cis to trans isomerization to prevent tau pathology in AD. Thus, during MCI and AD development, cis pThr231-Pro tau is the earliest detectable pathogenic tau conformation and antibodies and vaccines against the pathogenic cis p-tau may be used for the early diagnosis and treatment of AD. These findings offer in vivo approach to study conformational regulation of Pro-directed phosphorylation signaling.     

A blue-green absorbing cross-linker for rapid photoswitching of peptide helix content

Azobenzene derivatives can be used to reversibly photoregulate secondary structure when introduced as intramolecular bridges in peptides and proteins. Here we report the design, synthesis, and characterization of a disubstituted N,N-dialkyl azobenzene derivative that absorbs near 480 nm in aqueous solution and relaxes with a half-life of approximately 50 ms at room temperature. The wavelength of maximum absorbance and the rate of thermal relaxation are solvent-dependent. An increase in the percentage of organic solvent leads, in general, to a blue shift in the absorbance maximum and a slowing of the relaxation rate. In accordance with the design, the thermal relaxation of the azobenzene cross-linker from cis to trans causes an increase in the helix content of one peptide where the linker is attached via cysteine residues spaced at i, i + 11 positions and a decrease in helix content of another peptide with cysteine residues spaced at i, i + 7. This cross-linker design thus expands the possibilities for fast photocontrol of peptide and protein structure.