Helix Conformational Changes Research Articles

Detecting Structural Changes in Myosin using Bifunctional Spin Labels

Actomyosin interaction is responsible for driving a vast number of cellular processes, including force generation during muscle contraction. In this context, myosin is the molecular “motor” that produces muscle contraction. The myosin motor domain is comprised from three major subdomains: the force-generating domain (relay helix, SH-1, and converter), the ATP-binding pocket and the actin-binding cleft. During the cycle of force generation, myosin undergoes a series of structural rearrangements including “bending” of the relay helix, which communicates the biochemical state of the ATP binding pocket to the lever arm. The relay helix has previously been shown to reflect changes in distance, dynamics and orientation by electron paramagnetic resonance (EPR) with nucleotide state of the myosin catalytic domain. We now observe relay helix conformational changes using two rigidly attached bifunctional spin labels (BSL) on the relay helix (492.496) and in the lower 50 kDa domain (639.643) of the Dictyostelium myosin II motor domain by double electron electron resonance spectroscopy (DEER). We observe changes in relay helix conformation with various allosteric effectors - including potent myosin II inhibitors and physiological inhibitors such as site-directed oxidation. This approach allows for characterization of perturbations in chemo-mechanical coupling within the actomyosin complex under inhibitory conditions, as well as showing the reliability of relay helix spin probe sensitivity to the changes in myosin structural dynamics.

New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5′-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.

Shifting hydrogen bonds may produce flexible transmembrane helices

The intricate functions of membrane proteins would not be possible without bends or breaks that are remarkably common in transmembrane helices. The frequent helix distortions are nevertheless surprising because backbone hydrogen bonds should be strong in an apolar membrane, potentially rigidifying helices. It is therefore mysterious how distortions can be generated by the evolutionary currency of random point mutations. Here we show that we can engineer a transition between distinct distorted helix conformations in bacteriorhodopsin with a single-point mutation. Moreover, we estimate the energetic cost of the conformational transitions to be smaller than 1kcal/mol. We propose that the low energy of distortion is explained in part by the shifting of backbone hydrogen bonding partners. Consistent with this view, extensive backbone hydrogen bond shifts occur during helix conformational changes that accompany functional cycles. Our results explain how evolution has been able to liberally exploit transmembrane helix bending for the optimization of membrane protein structure, function, and dynamics.

Conformational Structural Changes of Bacteriorhodopsin Adsorbed onto Single-Walled Carbon Nanotubes

The interaction between purple membranes, composed of proteins of bacteriorhodopsin (bR) and their native surrounding lipids, and single-walled carbon nanotubes (SWNT) has been investigated. In this work, sonication has been used to debundle SWNT in buffer solution without surfactant before the addition of native purple membranes. The sample was then sonicated in a bath for a short time, followed by a centrifugation. The supernatants contain proteins in excess and SWNT as individual and small bundles covered by a bR layer with an average thickness of 1.5 nm. TEM and AFM observations support the idea that only a protein monolayer surrounds the tubes. Optical absorption and infrared spectroscopy measurements provide evidence that the proteins adsorbed onto the SWNT undergo orientational changes of the helical segments in bR and helix conformational changes. We ascribe the main driving force to the hydrophobic interactions between the sidewall of the SWNT and the hydrophobic residues of the alpha-helices of bR.

Probing the effect of side chains on the conformation and stability of helical peptides via isotope-edited infrared spectroscopy.

The mechanism of helix stabilization or destabilization by different amino acids has been the subject of several experimental and theoretical studies; these studies suggest that large or bulky side chains may modulate helix stability by altering the hydration of the helix backbone. In this paper, we report a spectroscopic study to determine the effect of alanine to leucine substitutions on the conformation and solvation of specific segments of a model helical peptide. A 25-residue, alanine-rich, helical peptide [Ac-(AAAAK)(4)-AAAAY-NH(2) (AKA)] and its two leucine variants [Ac-LLLLK-(AAAAK)(3)-AAAAY-NH(2) (LKA) and Ac-(AAAAK)(4)-LLLLY-NH(2) (AKL)] were characterized by infrared (IR) and electronic circular dichroism (ECD) spectroscopies. Introduction of (13)C isotopes into specific, consecutive, backbone carbonyls for certain blocks of each of the peptides mentioned above allows the IR spectra to be interpreted in terms of the conformation and solvation of specific residues within the helix. These isotope-edited IR spectra of the leucine peptides do not show evidence of a decrease in the degree of backbone solvation compared to the alanines, but suggest that the peptide may adopt a distorted conformation to accommodate the larger leucine side chains at the N-terminus. These experiments demonstrate the power of isotope-edited IR in dissecting subtle changes in helix conformation at the residue level.

DNA Duplexes Containing Photoactive Derivatives of 2′-Deoxyuridine as Photocrosslinking Probes for EcoRII DNA Methyltransferase-Substrate Interaction

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5′…CC*T/AGG…3′ and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2′-deoxyuridine (i5dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2′-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRH recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide- peptides obtained. DNA duplexes containing i5dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly268-Met391 region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.

Structural Basis for a Change in Substrate Specificity: Crystal Structure of S113E Isocitrate Dehydrogenase in a Complex with Isopropylmalate, Mg2+, and NADP,

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.

Synthesis and characterization of novel palladium(II) complexes of bis(thiosemicarbazone). Structure, cytotoxic activity and DNA binding of Pd(II)-benzyl bis(thiosemicarbazonate)

The preparation of palladium(II) complexes of 3,5-diacyl-1,2,4-triazole bis(thiosemicarbazone) (H 2L 2), 2,6-diacylpyridine bis(thiosemicarbazone) (H 2L 3) and benzyl bis(thiosemicarbazone) (H 2L 4) is described. The new complexes [PdCl 2(H 2L 2)] ( 1), [PdCl 2(H 2L 3)] ( 2) and [PdL 4] ·DMF ( 3) have been characterized by elemental analyses and spectroscopic studies (IR, 1H NMR and UV–Vis). The crystal and molecular structure of PdL 4 ·DMF (L=bideprotonated form of benzyl bis(thiosemicarbazone)) has been determined by single-crystal X-ray diffraction: green triclinic crystal, a=10.258(5), b=10.595(5), c=11.189(5) Å, α=97.820(5), β=108.140(5), γ=105.283(5)°, space group P1, Z=1. The palladium atom is tetracoordinated by four donor atoms (SNNS) from L 4 to form a planar tricyclic ligating system. The testing of the cytotoxic activity of compound 3 against several human, monkey and murine cell lines sensitive (HeLa, Vero and Pam 212) and resistant to cis-DDP (Pam- ras) suggests that compound 3 might be endowed with important antitumor properties since it shows IC 50 values in a μM range similar to those of cis-DDP [ cis-diamminedichloroplatinum (II)]. Moreover, compound 3 displays notable cytotoxic activity in Pam- ras cells resistant to cis-DDP (IC 50 values of 78 μM versus 156 μM, respectively). On the other hand, the analysis of the interaction of this novel Pd-thiosemicarbazone compound with DNA secondary structure by means of circular dichroism spectroscopy indicates that it induces on the double helix conformational changes different from those induced by cis-DDP.

Photomodulation of polypeptide conformation by sunlight in spiropyran-containing poly(L-glutamic acid)

Photochromic vinyl polymers, such as polyacrylates containing spiropyran groups, were found to undergo photoinduced variations of their viscosity. Since the viscosity of a polymer system is in part a reflection of polymer conformation, the photoviscosity effects were generically attributed to photoinduced conformational changes of the macromolecules. From the point of view of conformational properties, photochromic polypeptides are much more attractive systems, since they can exist in definite ordered structures such as {alpha}-helix or {beta}-structures, and their conformational variations can be directly investigated by means of CD measurements. In addition, their structure is much more relevant to the proteic nature of biological photoreceptors. In this context the authors report the first preparation of a photoresponsive polypeptide containing spiropyran units in the side chains and clear CD evidence that the polypeptide can undergo large random coil {r equilibrium} {alpha}-helix conformational changes upon exposure to sunlight and dark conditions, alternately. Moreover, irradiation in solvent mixtures having appropriate compositions allows the extent of the photoresponse to be controlled.

Topoisomer gel retardation: detection of anti-Z-DNA antibodies bound to Z-DNA within supercoiled DNA minicircles.

Small DNA fragments of approximately 350 bp in length, either with or without d(CG)n tracts, are ligated into underwound DNA minicircles to generate topoisomeric rings with different topological linking numbers, Lk. These minicircles, differing by an Lk of one, can be separated by acrylamide gel electrophoresis. Furthermore, electrophoresis can be used to reveal DNA double helix conformational changes that are induced by supercoiling, such as left-handed Z-DNA. When anti-Z-DNA antibodies are added to such minicircles, their binding leads to a selective retardation of the electrophoretic migration of the Z-DNA containing circles. This effect is not seen with relaxed minicircles and those with insufficient torsional stress to induce a conformational transition. Thus the technique of 'topoisomer gel retardation' presents a very sensitive assay for the identification of proteins that selectively bind to DNA conformations stabilized by negative DNA supercoiling.