Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α‐helix. The N‐terminus of Orai proteins contains a conserved CaM‐binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single‐molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N‐termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.     

Allosterically Activated Protein Self‐Assembly for the Construction of Helical Microfilaments with Tunable Helicity

Protein allostery, a chemical‐to‐mechanical effect that can precisely regulate protein structure, exists in many proteins. Herein, we demonstrate that protein allostery can be used to drive self‐assembly for the construction of tunable protein architectures. Calmodulin (CaM) was chosen as a model allosteric protein. Ca²⁺‐mediated contraction of CaM to a closed state can activate CaM and its ligand to self‐assemble into a 1D protein helical microfilament. Conversely, relaxation of CaM to the open state can unwind and further dissociate the helical assemblies. Fine regulation of the protein conformation by tuning the external Ca²⁺ level allows us to obtain various protein helical nanostructures with tunable helicity. This study offers a new approach toward chemomechanically controlled protein self‐assembly.     

Binding studies of nNOS-active amphibian peptides and Ca2+ calmodulin, using negative ion electrospray ionisation mass spectrometry

Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure.     

Using nitrile-derivatized amino acids as infrared probes of local environment

It is well-known that the C=N stretching vibration in acetonitrile is sensitive to solvent. Therefore, we proposed in this contribution to use this vibrational mode to report local environment of a particular amino acid in proteins or local environmental changes upon binding or folding. We have studied the solvent-induced frequency shift of two nitrile-derivatized amino acids, which are, AlaCN and PheCN, in H(2)O and tetrahydrofuran (THF), respectively. Here, THF was used to approximate a protein's hydrophobic interior because of its low dielectric constant. As expected, the C=N stretching vibrations of both AlaCN and PheCN shift as much as approximately 10 cm(-1) toward higher frequency when THF was replaced with H2O, indicative of the sensitivity of this vibration to solvation. To further test the utility of nitrile-derivatized amino acids as probes of the environment within a peptide, we have studied the binding between calmodulin (CaM) and a peptide from the CaM binding domain of skeletal muscle myosin light chain kinase (MLCK(579-595)), which contains a single PheCN. MLCK(579-595) binds to CaM in a helical conformation. When the PheCN was substituted on the polar side of the helix, which was partially exposed to water, the C=N stretching vibration is similar to that of PheCN in water. In constrast, when PheCN is introduced at a site that becomes buried in the interior of the protein, the C=N stretch is similar to that of PheCN in THF. Together, these results suggest that the C=N stretching vibration of nitrile-derivatized amino acids can indeed be used as local internal environmental markers, especially for protein conformational studies.     

The ATCUN domain as a probe of intermolecular interactions: application to calmodulin-peptide complexes

The utility of a three-residue Cu2+ binding motif (ATCUN domain) for studying intermolecular interactions is demonstrated. By comparing a set of 1H-15N correlation spectra recorded on complexes of calmodulin (CaM) and peptides with the ATCUN tag in the presence and absence of Cu2+ the two possible canonical binding orientations of the peptide can be rapidly distinguished. The methodology is confirmed with studies of complexes of CaM and peptides from myosin light chain kinase and CaM kinase kinase, for which high-resolution structures are available, and then applied to a complex with CaM kinase I for which structural data has not been obtained. The orientation of the CaM kinase I and myosin light chain kinase peptides are shown to be identical. In the case of a complex of CaM with a peptide for which structural information is not available, the present methodology, in combination with 1H-15N residual dipolar couplings measured on CaM, and the database of existing CaM-peptide structures, allows a homology model to be built rapidly and with confidence.     

Structural insights into calmodulin/adenylyl cyclase 8 interaction

Calmodulin (CaM) is a highly conserved intracellular Ca²⁺-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca²⁺-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca²⁺ sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

Noncovalent domino effect on helical screw sense of chiral peptides possessing C-terminal chiral residue

Recently, a novel chiral intermolecular interaction was found in an N-deprotected achiral nonapeptide that undergoes the predominance of one-handed screw sense through the addition of chiral small carboxylic acid (Inai, Y.; Tagawa, K.; Takasu, A.; Hirabayashi, T.; Oshikawa, T.; Yamashita, M. J. Am. Chem. Soc. 2000, 122, 11731). We here clarify to what extent such noncovalent chiral domino effect affects the helical screw sense of an N-deprotected chiral peptide. Two chiral peptides consisting of C-terminal L-Leu (1) or L-Leu(2) (2) and the preceding achiral helical octapeptide segment were employed. NMR and IR spectroscopy, and energy calculation indicated that both peptides adopt a helical conformation in chloroform. Peptide 1 showed a small excess of a left-handed screw sense for the achiral helical octapeptide, but peptide 2 strongly preferred a right-handed screw sense. The addition of chiral Boc amino acid to a chloroform solution of peptide 1, depending on its chirality, underwent a unique helix-to-helix transition or led to remarkable stabilization of the original left-handed screw sense. Peptide 2 retained the original right-handed screw sense on addition of chiral Boc-amino acid, but its helical stability changed to some extent depending on its added chirality. Therefore, the importance of noncovalent domino effect for controlling the helical screw sense or helical stability of a chiral peptide has been demonstrated here for the first time. In addition, we here have presented a unique system that both N-terminal noncovalent and C-terminal covalent domino effects operate simultaneously on the helical screw sense of a single achiral segment and have compared both powers for inducing the screw sense bias.     

Revealing two-state protein-protein interactions of calmodulin by single-molecule spectroscopy

We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.     

Transient, sparsely populated compact states of apo and calcium-loaded calmodulin probed by paramagnetic relaxation enhancement: interplay of conformational selection and induced fit

Calmodulin (CaM) is the universal calcium sensor in eukaryotes, regulating the function of numerous proteins. Crystallography and NMR show that free CaM-4Ca(2+) exists in an extended conformation with significant interdomain separation, but clamps down upon target peptides to form a highly compact structure. NMR has revealed substantial interdomain motions in CaM-4Ca(2+), enabled by a flexible linker. In one instance, CaM-4Ca(2+) has been crystallized in a compact configuration; however, no direct evidence for transient interdomain contacts has been observed in solution, and little is known about how large-scale interdomain motions contribute to biological function. Here, we use paramagnetic relaxation enhancement (PRE) to characterize transient compact states of free CaM that are too sparsely populated to observe by traditional NMR methods. We show that unbound CaM samples a range of compact structures, populated at 5-10%, and that Ca(2+) dramatically alters the distribution of these configurations in favor of states resembling the peptide-bound structure. In the absence of Ca(2+), the target peptide binds only to the C-terminal domain, and the distribution of compact states is similar with and without peptide. These data suggest an alternative pathway of CaM action in which CaM remains associated with its kinase targets even in the resting state. Only CaM-4Ca(2+), however, shows an innate propensity to form the physiologically active compact structures, suggesting that Ca(2+) activates CaM not only through local structural changes within each domain but also through more global remodeling of interdomain interactions. Thus, these findings illustrate the subtle interplay between conformational selection and induced fit.     

The kinetics of helix unfolding of an azobenzene cross-linked peptide probed by nanosecond time-resolved optical rotatory dispersion

The unfolding dynamics of a 16 amino acid peptide (Ac-EACAREAAAREAACRQ-NH(2), FK-11-X) was followed using nanosecond time-resolved optical rotatory dispersion (ORD). The peptide was coupled to an azobenzene linker that undergoes subnanosecond photoisomerization and reisomerizes on a time scale of minutes. When the linker is in the trans form, the peptide favors a more helical structure (66% helix/34% disordered) and when in the cis configuration the helical content is reduced. Unfolding of FK-11-X was rapidly triggered by a 7-ns laser pulse at 355 nm, forming cis azobenzene-linked peptides that maintained the secondary structure (helical or disordered) of their trans azobenzene counterparts. The incompatibility of the instantaneous cis photoproduct with helical secondary structure drives the subsequent peptide unfolding to a new conformational equilibrium between cis helix and cis disordered structures. The kinetic results show a approximately 40% decrease in the time-dependent ORD signal at 230 nm that is best fit to a single-exponential decay with a time constant of 55 +/- 6 ns. Folding and unfolding rates for cis FK-11-X are estimated to be approximately 3.0 x 10(6) s(-)(1) (1/330 ns) and approximately 1.5 x 10(7) s(-)(1) (1/66 ns), respectively.     

Determining the topology of integral membrane peptides using EPR spectroscopy

This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), was attached to the pore-lining transmembrane domain (M2delta) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14 degrees was calculated for the M2delta peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000-fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 microg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.     

影响多肽与花粉钙调素亲和性的因素──多肽的圆二色性及核磁共振研究

钙调素 ( Ca M)存在于所有真核细胞生物体内 ,它可与很多天然的生物活性肽结合 ,如 β-内啡肽、胰高血糖素和某些昆虫毒液肽等 [1] .有关 Ca M与多肽相互作用的研究普遍认为 ,对 Ca M有高亲和性的多肽应该具有形成α螺旋结构的显著倾向[2 ] .为进一步确认多肽的主链构象对 Ca M亲和能力的影响 ,我们采用圆二色性光谱和核磁共振波谱分析了荞麦花粉碱性十二肽 BPP-1和它的类似物 BPP-3的结构特征 ,配合多肽对花粉钙调素 ( p Ca M)的结合能力 ,发现肽链的可塑性和 C端的极性是影响多肽与 p Ca M亲和能力的因素 ,而形成 α螺旋结构的倾…     

Chiroptical transcription of helical information through supramolecular harmonization with dynamic helices

With biologically important "peptide bundling" as the motif, new chromophoric cyclic host 1 was designed, which consists of two zinc porphyrin units that are connected by dynamic peptide helices of nonameric aminoisobutyric acid (Aib) units. Upon inclusion of pyridine-anchored helical peptides between the zinc porphyrin units, 1 displayed an intense exciton-coupled circular dichroism (CD) band at 410-450 nm, whose sign reflected the helical sense of the guest peptides. Studies with conformationally defined dehydrophenylalanine-containing analogues indicated that the dynamic helical chains in the host are stereochemically harmonized with right- or left-handed helices of the guest peptides in a confined nano space, leading to either clockwise- or anticlockwise-twisted geometry (chiroptical output) of the connecting zinc porphyrin chromophores.     

Probing the role of the C-H...O hydrogen bond stabilized polypeptide chain reversal at the C-terminus of designed peptide helices. Structural characterization of three decapeptides

The structural characterization in crystals of three designed decapeptides containing a double d-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all l analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed alpha-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with (D)Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-H.O hydrogen bond between residue 4 C(alpha)H and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C(alpha) atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt alpha(L) conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-H.O hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures.     

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

β Helices—helices formed by alternating d,l ‐peptides and stabilized by β‐sheet hydrogen bonding—are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of β‐helical structure by presenting the first design and biophysical characterization of a hydrophilic d,l ‐peptide, 1 , that forms a β helix in methanol. The design of 1 is based on the β‐hairpin/β helix—a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected β‐hairpin/β‐helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR‐based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol ( 1 m ) transitions cooperatively to an extended conformation ( 1 w ) resembling a cyclic β‐hairpin; observation of an isodichroic point in the solvent‐dependent CD spectra indicates that this transition is a two‐state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 °C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the ¹H NMR CαH spectroscopic resonances of 1 m are downfield‐shifted with respect to random‐coil values, a hitherto unreported property for β helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed β‐helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water‐soluble β helices.     

Protein folding in a reverse micelle environment: the role of confinement and dehydration

Characterization of the molecular interactions that stabilize the folded state of proteins including hydrogen bond formation, solvation, molecular crowding, and interaction with membrane environments is a fundamental goal of theoretical biophysics. Inspired by recent experimental studies by Gai and co-workers, we have used molecular dynamics simulations to explore the structure and dynamics of the alanine-rich AKA(2) peptide in bulk solution and in a reverse micelle environment. The simulated structure of the reverse micelle shows substantial deviations from a spherical geometry. The AKA(2) peptide is observed to (1) remain in a helical conformation within a spherically constrained reverse micelle and (2) partially unfold when simulated in an unconstrained reverse micelle environment, in agreement with experiment. While aqueous solvation is found to stabilize the N- and C-termini random coil portions of the peptide, the helical core region is stabilized by significant interaction between the nonpolar surface of the helix and the aliphatic chains of the AOT surfactant. The results suggest an important role for nonpolar peptide-surfactant and peptide-lipid interactions in stabilizing helical geometries of peptides in reverse micelle environments.     

Demonstration of α-helical structure of peptides tethered to gold surfaces using surface infrared and circular dichroic spectroscopies

Gold and quartz surfaces terminated in an alkane thiol self-assembled monolayer (SAM) that were partially terminated with azide were reacted with a helical peptide containing two alkyne groups in a Cu(I)-catalyzed Huisgen cycloaddition. Surface grazing incidence angle reflection-absorption infrared spectroscopy (GRAS-IR) was used to determine that when the Au surface was terminated with 25% of the monolayer containing azide groups, 92% of available azide groups reacted with the peptide. The majority of peptides reacted with both alkynes, resulting in peptides tethered to the surface through two covalent bonds. This was confirmed by comparison to a control peptide containing only one reactive alkyne group. Surface circular dichroic (CD) spectroscopy showed that while the helical structure of the peptide was distorted in the reaction solution, α-helical structure was induced when tethered on the SAM functionalized Au surface. Demonstration of the preservation of desired secondary structure of helical elements at a chemically functionalized surface is an important advance in preparing robust biologically mimetic surfaces to integrate functioning proteins into inorganic materials.     

A top-down LC-FTICR MS-based strategy for characterizing oxidized calmodulin in activated macrophages

A liquid chromatography-mass spectrometry (LC-MS)-based approach for characterizing the degree of nitration and oxidation of intact calmodulin (CaM) has been used to resolve ～250 CaM oxiforms using only 500 ng of protein. The analysis was based on high-resolution data of the intact CaM isoforms obtained by Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) coupled with an on-line reversed-phase LC separation. Tentative identifications of post-translational modifications (PTMs), such as oxidation or nitration, have been assigned by matching observed protein mass to a database containing all theoretically predicted oxidation products of CaM and verified through a combination of tryptic peptide information (generated from bottom-up analyses) and on-line collisionally induced dissociation (CID) tandem mass spectrometry (MS/MS) at the intact protein level. The reduction in abundance and diversity of oxidatively modified CaM (i.e., nitrated tyrosines and oxidized methionines) induced by macrophage activation has been explored and semiquantified for different oxidation degrees (i.e., no oxidation, moderate, and high oxidation). This work demonstrates the power of the top-down approach to identify and quantify hundreds of combinations of PTMs for single protein target such as CaM and implicate competing repair and peptidase activities to modulate cellular metabolism in response to oxidative stress.     

Monolayer formation of short helical turn forming peptide derivatives at the air–water and air–solid interfaces

Two tetrapeptide derivatives [peptide A (Boc–Ala–Ile–Ile–Gly–OMe) and peptide B (Boc–Ala–Ile–Leu–Ser–OMe)], that take helical turn conformation in solution, were shown to form monolayer at the air/water interface. Circular dichroism (CD) measurements indicate that peptide A has more helical turn propensity than peptide B in sodium dodecyl sulphate (SDS) micelles. Langmuir–Blodgget film study of peptides A and B suggest that both the peptides form stable monolayer at the air/water interface. Spectroscopic investigations reveal that peptide A forms helical turn assemblage on transferring the film into hydrophilic quartz and hydrophobic ZnSe surfaces. Whereas, peptide B adopts β-sheet structure on hydrophilic surface and a mixture of β-sheet and helical turn conformation on hydrophobic surface.     

α‐Peptide–Oligourea Chimeras: Stabilization of Short α‐Helices by Non‐Peptide Helical Foldamers

Short α‐peptides with less than 10 residues generally display a low propensity to nucleate stable helical conformations. While various strategies to stabilize peptide helices have been previously reported, the ability of non‐peptide helical foldamers to stabilize α‐helices when fused to short α‐peptide segments has not been investigated. Towards this end, structural investigations into a series of chimeric oligomers obtained by joining aliphatic oligoureas to the C‐ or N‐termini of α‐peptides are described. All chimeras were found to be fully helical, with as few as 2 (or 3) urea units sufficient to propagate an α‐helical conformation in the fused peptide segment. The remarkable compatibility of α‐peptides with oligoureas described here, along with the simplicity of the approach, highlights the potential of interfacing natural and non‐peptide backbones as a means to further control the behavior of α‐peptides.