Efficient Covalent Bond Formation in GasPhase Pep

Authors: Christopher J Shaffer Prokopis C Andrikopoulos Jan Řezáč Lubomír Rulíšek František Tureček

Publish Date: 2016/01/27

Volume: 27, Issue: 4, Pages: 633-645

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Abstract

Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine L tagged peptides GL n L m K n = 13 m = 20 were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm Carbene intermediates produced by photodissociative loss of N2 from the L diazirine rings underwent insertion into X−H bonds of the target peptide moiety forming covalent adducts with yields reaching 30 Gasphase sequencing of the covalent adducts revealed preferred bond formation at the Cterminal residue of the target peptide Siteselective carbene insertion was achieved by placing the L residue in different positions along the photopeptide chain and the residues in the target peptide undergoing carbene insertion were identified by gasphase ion sequencing that was aided by specific 13C labeling Density functional theory calculations indicated that noncovalent binding to GLLLK resulted in substantial changes of the GLLLK + H+ ground state conformation The peptide moieties in GLLLK + GLLLK + H+ ion complexes were held together by hydrogen bonds whereas dispersion interactions of the nonpolar groups were only secondary in groundstate 0 K structures BornOppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple residuespecific contacts between the diazirine carbons and GLLLK residues The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes Diazirine photochemistry in combination with BornOppenheimer molecular dynamics is a promising tool for investigations of peptide–peptide ion interactions in the gas phaseNoncovalent protein–protein and protein–ligand interactions are the basis of molecular recognition of critical importance to many areas of biology In addition to Xray diffraction and spectroscopic methods of structure elucidation crosslinking and photoaffinity labeling have been the major chemical methods to study protein noncovalent interactions dating back to the pioneering studies of Westheimer et al 1 and Knowles and coworkers 2 in the 1960s Detection of noncovalent interactions relies on crosslinking by covalent bond formation between the protein and ligand at their contact sites upon chemical reaction or photolysis Photochemical crosslinking in particular utilizes highly reactive intermediates such as radicals nitrenes 2 or carbenes 3 produced by photolysis of a chemically stable chromophore that is incorporated into the protein or ligand 4 5 With the introduction of photolabile amino acid residues photoleucine and photomethionine it became possible to achieve photochemical crosslinking in complexes closely resembling native systems such as membrane protein 6 and protein–peptide complexes 7 Photoleucine L2amino44azipentanoic acid abbreviated as L contains a diazirine ring that is a specific chromophore absorbing at 350–370 nm where the native peptide chromophores are transparent and are not photoactivated Photon absorption causes N2 elimination creating a highly reactive singlet carbene that undergoes insertion into proximate X−H bonds X = C N O to form new covalent H−C−X bonds 8 Competing with insertion the carbene can undergo fast intramolecular isomerization by 12hydrogen shift to form a nonreactive olefin in the photoactive component 9 10 11 12 which can no longer covalently bind to the complex counterpart This side reaction provides an internal clock that limits the time scale for carbeneX–H bond interactions to within 10–7 s The photochemical crosslinking “stitching” can be readily detected by mass spectrometry and the position of the new covalent link can be located by tandem mass spectrometry sequencing of gasphase adduct ions 7 A substantial drawback of solution studies is the very low yields of crosslinked products that must be separated from the unmodified starting material and side products 7 13 14 15An entirely different approach to studying noncovalent protein–ligand and protein–protein complexes relies on mass spectrometry that has been used to study their stoichiometry 16 17 stability 18 19 20 and thermochemistry in the gas phase 21 22 Formation of covalent bonds in gasphase ion complexes has been reported for several systems that relied on collisioninduced chemical reactions between an anion and a cation in a complex that mimicked chemical methods used in solution 23 24 In addition covalent bond formation in gasphase ion complexes with 18crown6ether has been accomplished by collisioninduced dissociation of diazomalonate derivatives 25 or photodissociation of a diazirine labeled peptide 26 in which reactive carbenes played the role of reactive intermediates The singular example whereby photoactivation of a peptide ion led to amide bond formation was severely limited by indiscriminate regioselectivity use of vacuum ultraviolet light 157 nm and low yield 1 27 Herein we exploit diazirine chromophores that are synthetically incorporated into peptides to generate reactive carbene intermediates for covalent intermolecular bond formation in noncovalent peptide–peptide complexes in the gas phase This photoactivation scheme provides a unique opportunity for efficient bond formation as a tool for probing the conformational structure of gaseous peptide–peptide complex ions in the absence of surrounding solvent ions membranes or other interacting medium As the photoactive components we chose hydrophobic pentapeptides GLLLK GLLLK GLLLK and GLLLK The first of these provides multiple reactive centers to undergo X−H bond insertion to a peptide counterpart in a nonspecific manner The other three peptides provide sequencespecific reactive centers produced by photolysis As counterparts in the noncovalent complexes we employ hydrophobic peptides GLLLG and GLLLK The GLLLG sequence motif appears in several transmembrane proteins eg those of the claudine family where the Gly residues are flanked by additional hydrophobic residues Val Leu Ile and are immersed in the lipid membrane 28 The GLLLK sequence motif appears in over 100 proteins and the Cterminal Lys is often flanked by another polar residue Lys His 29 The size of the peptides used for this gasphase study was deliberately limited to allow us to complement experimental investigations of photostitching with a conformational trajectory analysis using BornOppenheimer dynamics at an augmented semiempirical level of quantum theory including dispersion interactions In this way both polar and dispersion interactions between the peptide components can be treated at the same level of theory and their role in affecting the complex’ stability and dynamics can be assessed We wish to illustrate that this new photostitching approach achieves high yields of covalent bond formation provides insight into the structure and dynamics of noncovalent peptidepeptide complexes and allows us to elucidate the nature and fine features of interactions between the peptide moietiesCollision induced dissociation CID mass spectra were measured on a modified LTQXL ETD linear ion trap LIT mass spectrometer ThermoElectron Fisher San Jose CA USA Peptide solutions 5–10 μM in 50/50/1 methanol/water/acetic acid were electrosprayed at 22–23 kV from a pulled fused silica capillary into an open microspray ion source described previously 32 33 Ion pair complexes were selected according to their m/z and stored within the linear anion trap MSn experiments were carried out by isolating the ions and exposing them to resonant collisional excitation or photoexcitation The collisional activation times were typically varied between 30 ms for preliminary analysis and 1000 ms to create times similar to photodissociation conditionsTo accomplish photodissociation of trapped ions in the LIT the LTQXL ETD mass spectrometer was modified as reported previously 26 34 35 Briefly the chemical ionization CI source for the anion production was modified by drilling a 1mm diameter hole into the insert block to provide a line of sight path to the LIT The backside vacuum gate to the CI source was replaced by an aluminum plate carrying a quartz window The irradiating light beam was produced by an EKSPLA NL 301 HT Altos Photonics Bozeman MT USA NdYAG laser operating at 20 Hz frequency with a 3–6 ns pulse width The laser is equipped with a third harmonics frequency generator producing a single 355 nm wavelength at 120 mJ/pulse peak power The typical light intensity used in the photodissociation experiments was 18 mJ/pulse The laser beam of 6mm diameter is aligned by mirrors and focused by a telescopic lens to pass the small aperture drilled in the CI source The laser beam diameter in the LIT is estimated at 3–4 mm to ensure overlap with the trapped ions Both the laser system and the LTQXL are set on an optical table for optimum alignment The laser was interfaced to the LTQ by LabView software National Instruments Austin TX USA that receives a signal from a TTL pulse on pin14 of the J1 connector on the LTQ console Laser pulses are triggered internally by the EKSPLA system but power is controlled for each pulse by commands from the LabView software The typical experimental set up consists of selecting the ion to be photodissociated and storing it in the LIT for a chosen time period For example 400ms storage time can accommodate up to seven laser pulses spaced by 50 ms This allows one to vary the number of pulses and determine the photodissociation kinetics Longer storage times of 3 s allowing 60 laser pulses are readily realized

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