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Folding and Unfolding of Helix-Turn-Helix Motifs in the Gas Phase

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Folding and Unfolding of Helix-Turn-Helix Motifs in the Gas Phase FOCUS:FROM MOBILITIES TO PROTEOMES Folding and Unfolding of Helix-Turn-Helix Motifs in the Gas Phase Lloyd W. Zilch, David T. Kaleta, Motoya Kohtani, Ranjani Krishnan, and Martin F. Jarrold Department of Chemistry, Indiana University, Bloomington, Indiana, USA Ion mobility measurements and molecular dynamic simulations have been performed for a series of peptides designed to have helix-turn-helix motifs. For peptides with two helical ϩ ϩ ϩ ϩ sections linked by a short loop region: AcA14KG3A14K 2H , AcA14KG5A14K 2H , ϩ ϩ ϩ ϩ ϭ ϭ ϭ AcA14KG7A14K 2H , and AcA14KSar3A14K 2H (Ac acetyl, A alanine, G glycine, Sar ϭ sarcosine and K ϭ lysine); a coiled-coil geometry with two anti-parallel helices is the lowest energy conformation. The helices uncouple and the coiled-coil unfolds as the temperature is raised. Equilibrium constants determined as a function of temperature yield enthalpy and entropy changes for the unfolding of the coiled-coil. The enthalpy and entropy changes depend on the length and nature of the loop region. For a peptide with three helical sections: protonated AcA14KG3A14KG3A14K; a coiled-coil bundle with three helices side-by-side is substantially less stable than a geometry with two helices in an antiparallel coiled-coil and the third helix collinear with one of the other two. (J Am Soc Mass Spectrom 2007, 18, 1239–1248) © 2007 American Society for Mass Spectrometry ϩ ϩ he function and activity of a protein is controlled AcA15K H helices are remarkably stable in the gas by its conformation. The conformation is deter- phase, and have been shown to remain intact to over ϩ ϩ Tmined by the protein’s potential energy surface, 700 K. The two helices in the AcA14KG3A14K 2H which is in turn determined by the amino acid se- peptide are separated by a G3 loop. Glycine was used to quence. We often think of the structure of a folded make the loop because it is a helix breaker, i.e., it has protein in a hierarchical way starting with the second- low propensity to form helices in solution [6] and in the ϩ ϩ ary structure elements and how they aggregate into gas phase [7, 8]. The AcA14KG3A14K 2H peptide is motifs. The motifs then provide the building blocks for thus expected to possess two main conformations, at the larger structures or domains which provide the low temperature a coiled-coil arrangement with the two protein with its three dimensional structure [1]. The helices aligned side-by-side, and at high temperature an process of protein folding often appears to follow a unfolded conformation where the helices are uncoupled similar hierarchical pathway, where in many cases the [4]. In the coiled-coil geometry the helices are aligned secondary structure elements appear early along the antiparallel. This permits favorable interactions be- folding pathway. tween the helix dipoles, however, the antiparallel ar- The helix-turn-helix is one of the simplest and most rangement is less common in nature than the parallel commonly occurring motifs in nature. It is found in a coiled-coil [9]. variety of situations, including the DNA binding [2] In this paper, we report studies of several peptides and calcium binding [3] motifs. Previously, we have with the helix-turn-helix motif. In particular, we focus reported preliminary studies of the peptide on a series of peptides with turns or loop sections of ϭ ϭ ϭ AcA14KG3A14K (G3) (Ac acetyl, A alanine, G different lengths and composition. In AcA14KSar3A14K glycine, and K ϭ lysine) that was designed to adopt a (Sar3) sarcosine substitutes for the glycine loop in ϩ helix-turn-helix motif in the gas phase [4]. In the 2 AcA14KG3A14K. Sarcosine is a glycine derivative with a charge state of this peptide, both lysines are protonated, methyl group replacing a hydrogen atom on the amide and the A14K units are expected to be helical. The nitogen, so that this residue cannot form helical hydro- protonated lysine side-chain caps the C-terminus of the gen bonds with the amide nitrogen. In AcA14KG5A14K helix, and in this configuration, the charge interacts (G5) and AcA14KG7A14K (G7) the length of the glycine favorably with the helix dipole [5]. Isolated loop is increased to five and seven residues respec- tively. Finally, AcA14KG3A14KG3A14K (3HG3) has three A14K helical segments separated by G3 loops. The Address reprint requests to Dr. M. F. Jarrold, Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN 47405-7102, question with this peptide is whether or not the third USA. E-mail: [email protected] helix will fold into a three helix coiled-coil bundle. This Published online April 13, 2007 © 2007 American Society for Mass Spectrometry. Published by Elsevier Inc. Received January 2, 2007 1044-0305/07/$32.00 Revised March 28, 2007 doi:10.1016/j.jasms.2007.03.027 Accepted March 29, 2007 1240 ZILCH ET AL. J Am Soc Mass Spectrom 2007, 18, 1239–1248 arrangement occurs naturally, for example, in the spec- by microprocessor based temperature controllers that trin repeat [10]. operate cryovalves that are fed from a liquid nitrogen The objective of these studies is to learn about the tank. The high temperature drift tube is heated by helix-turn-helix motif using simple model peptides in a Thermocoax heaters (Alpharette, GA) connected to a solvent-free environment. There are two reasons why variable voltage transformers. The temperature is reg- studies of unsolvated peptides are desirable. First, by ulated using microprocessor based controllers. removing the interactions with the solvent, the system Some of the ions that travel across the drift tube exit becomes much simpler to model and understand; and through a small aperture. These ions are focused into a second, the aqueous environment is not the only envi- quadrupole mass spectrometer set to transmit the ion of ronment that is important from a biological point of interest. Ions that are transmitted through the quadru- view. Membrane proteins are a large class of proteins pole are detected by an off-axis collision dynode and that are involved in transport, recognition, and ligand- dual microchannel plates. Average collision cross sec- receptor binding. A large portion of the surface of tions are determined by using an electrostatic shutter at membrane proteins is shielded from water. Even glob- the entrance of the drift tube to admit short pulses of ular proteins with hydrophilic surfaces have a hydro- ions; the ion signal at the detector is then recorded with phobic core from which water is largely excluded and a multichannel scaler synchronized with the electro- interactions occur in a water-free environment. static shutter. Drift time distributions are obtained by In the studies described here, ion mobility measure- correcting the measured arrival time distributions to ments [11, 12] in conjunction with molecular dynamics account for the time that the ions spend traveling simulations are used to investigate the conformations of outside of the drift tube. The measured drift times are the helix-turn-helix peptides as a function of tempera- converted into collision cross sections using: ture. In the ion mobility measurements the ions move through a buffer gas under the influence of a weak 1⁄2 (18␲)1⁄2 1 1 ze t E electric field. How rapidly they travel depends on the ⍀1,1 ϭ ͫ ϩ ͬ D avg 1⁄2 average collision cross section: ions with unfolded 16 m mb (kBT) Lp structures have larger collision cross sections, and move through the buffer gas more slowly than ions with [14] where m and mb are the masses of the ion and compact, roughly-spherical structures. Conformations buffer gas, respectively, ze is the charge, tD is the drift are assigned by comparing the measured cross sections time, E is the drift field, and ␳ is the buffer gas number to average cross sections calculated for structures de- density. rived from molecular dynamics simulations. In the All peptides were synthesized using FastMoc chem- present work, the two main classes of conformations istry on the Applied Biosystems Model 433A peptide expected here (the coiled-coil with anti-parallel helices synthesizer (Foster City, CA). Normal FastMoc proce- and the unfolded conformation where the helices are dures were used for G3, Sar3, G5, and G7. A special uncoupled) are easily distinguished on the basis of their procedure was used for the synthesis of the peptide mobilities. with three helices, 3HG3. N-[(Dimethylamino)-1H- 1,2,3,-triazolo[4,5-b]pyridin-1-ylmethylene]-N- methylmethanaminium Hexafluorophosphate N-oxide Experimental (HATU) was used for activation, and 10% 1,8-diazabicyclo Ion mobility measurements were performed as a func- [5,4,0]-undec-7-ene, 10% piperidine in DMF was used tion of temperature to obtain information about the for deprotection. This mixture was used to disrupt conformations present for the G3, G5, G7, Sar3, and ␤-sheet formation on the HMP resin, which increasingly 3HG3 peptides. The experimental apparatus used for hinders coupling at longer polymer lengths. After these studies has been described in detail previously cleavage with a 95% trifluoroacetic acid/5% water [13, 14]. Briefly, peptide solutions were electrosprayed solution, the peptides were precipitated with diethyl in air, and the ions enter the apparatus through a heated ether, centrifuged, and lyophilized. The peptides were capillary interface. The ions pass through a differen- used unpurified, but MALDI-TOF was used to verify tially pumped region, through an RF hexapole ion the product. Electrospray solution consisted of 2 mg of guide, and they are then focused into the drift tube. The peptide and 1 mL of TFA with 0.1 mL water in all cases. drift tube is operated with around 4 torr of helium buffer gas and with a drift voltage of 180 to 380 V.
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