AN EXPERIMENTAL & THEORETICAL STUDY OF ALKALI CATION/SULFUR CONTAINING AMINO ACID INTERACTIONS Amy Gabriel, Bob Moision, & Peter B. Armentrout Department of Chemistry University of Utah Salt Lake City UT 84112

Introduction Methionine Peptide and protein sequencing using a mass spectrometer is quickly becoming a practical technique. One common sequencing method is to fragment protonated peptides in the gas phase using collision induced dissociation (CID), and then to use the identity of the fragments to deduce the original amino acid sequence. A potential problem with the use of many protonated peptides is that protonation may occur at a number of sites on the peptide; and this, in turn, can lead to unpredictable fragmentation. More predictable fragmentation may be possible by placing a localized charge in the form of a metal ion onto the peptide. Here, we examine alkali metal (lithium, sodium, & potassium) cation binding with a sulfur containing amino acid, methionine. This work is important in providing fundamental binding interactions and key insights of the reaction thermodynamics specific to amino acids, peptides, and ultimately, proteins. The determination of the bond dissociation energies (BDE) of the metal-ligand complexes is done experimentally by collision induced dissociation of the complexes with xenon using guided ion beam mass spectrometry; as well as, theoretically. Methionine

CID: Collision Induced Dissociation An accurate method for determining the strength of a metal ion (M+)-neutral ligand (L) bond. The metal-ligand complex is collided with xenon at a well defined and variable kinetic energy. With the “right” amount of energy the metal- ligand bond will break causing the metal ion to dissociate. The fragmentation products are detected as a function of the collision energy, thus determining the threshold energy, Eo, of the metal-ligand dissociation.

THE GIBMS: Guided Ion Beam Mass Spectrometer He & Ar inlet 1 2 1. Metal ions are produced in a DC discharge by Ar sputtering the alkali metal of interest. The neutral ligand is introduced using a customized heater. Metal-ligand complexes are formed in the 1 m long flow tube via associative reactions. The complexes are thermalized by ~105 collisions with the He buffer gas. 2. After travelling through a numberof electrostatic lenses the reactant ion is focused into a magnetic momentum analyzer which is tuned to select a single species for further analysis. 3 4 3. The reactant ion is brought into an rf octopole ion guide at a well defined kinetic energy and enters the collision cell which contains a gas, usually xenon. At higher energies, collisions can cause fragmentation of the reactant ion into product ions. 4. Any unreacted parent and product ions are separated in the quadropole mass selector, then counted using the ion detector, and the intensities recorded.

Consider... To accurately model and extract the true threshold energy from laboratory data The reactant ions are thermalized at 300K in the flow tube. The reactants, therefore, already have finite internal energy in vibrations and rotations before the collision with xenon. ab initio theory is used to determine the vibrational frequencies and rotational constants of the complex and neutral ligand. Multiple collisions will cause a erroneously low threshold energy, thus metal-ligand complex collision data is taken at three xenon pressures. The results are extrapolated to zero pressure to ensure that only single collision conditions are modeled. Given an infinite amount of time for dissociation an energized molecule (E > Eo) will dissociate into the products. However, in the GIBMS the energized ions have a finite amount of time for detection. Statistical RRKM theory is used to model this lifetime effect.

CID of MetNa+

Searching for Structures... The low energy structures of each metal-ligand complex & neutral species need to be determined for the calculation of theoretical binding energies. Even for a simple single amino acid system, many possible conformations exist. A simulated annealing protocol using the AMBER forcefield was used to search for possible structures. AMBER can quickly generate a large number of possible structures. Unique structures are then submitted for energy minimization at a low ab initio level (hf/3-21G). The hf/3-21G energies were used to intelligently select structures for higher level ab initio optimizations, frequency calculations, and single point energies. All theoretical results reported here include zero point energy (ZPE) corrections. Calculations at the mp2(full)/6-311+G(2d, 2p) level also include corrections for basis set superposition error (BSSE).

MetNa+ Structures mp2(full)/6-311+G(2d, 2p)//b3lyp/6-311G** with ZPE ~8 kJ/mol 0 kJ/mol The low energy structures of MetNa+ are clearly tridentate to S, N, and O. Interestingly, subtle changes in the side chain orientation results in noticeable changes in energy. ~27 kJ/mol

MetK+ Structures mp2(full)/6-311+G(2d, 2p)b3lyp/6-311G** with ZPE 0 kJ/mol ~ 7 kJ/mol ~ 3 kJ/mol Like Na+, K+ binds Met tridentate through the S, N, and O. However, MetK+ also has a low energy conformation where the K+ binds to both of the O’s and S. Analogous structures for MetNa+ are ~33 kJ/mol higher relative to the low energy MetNa+ conformer.

CID of MetK+

MetLi+ Structures mp2(full)/6-311+G(2d, 2p)//b3lyp/6-311G** with ZPE ~5 kJ All low energy structures for MetLi+ are bound tridentate through the S, N, and O to Li+ in the same fashion as Na+. Li+ forms a larger number of tridentate structures relative to Na+ and K+. 0 kJ ~12 kJ ~11 kJ

CID of MetLi+

Bond Dissociation Energies (eV)

Results Li+ Na+ K+

Results

Conclusions & Future Work Agreement between theoretical & experimental BDEs is, in general, quite good. All levels of theory examined here tend to underestimate the MetLi+ BDE. At the mp2(full)/6-311+G(2d, 2p) level exclusion of BSSE results in slightly better agreement with experimental results. Low energy structures for all three alkali metals show tridentate binding to the amine, carboxylic acid carbonyl oxygen, and the side chain sulfur. Unlike Li+ and Na+, K+ has low energy structures where binding is to both carboxylic acid oxygens and the sulfur. Subtle changes to the side chain orientation of the Met make noticeable changes to the energy. Future work will include examination of the cysteine, the other sulfur containing amino acid, and higher level calculations on the methionine complexes studied here.

Low Energy Cysteine Structures ~0 kJ ~0 kJ CysK+ CysLi+ ~11 kJ ~3 kJ

Lithium, Sodium, & Potassium In general these elements are S-block elements called alkali metal with one valence electron in their outer shell. They are hard cations that favor nitrogen or oxygen donor ligands like amino acids. These alkali metals form generally poor complexes, but do bind weakly to chelating ligands and proteins providing substrate links to the enzyme for orientation purposes. Li+ is a biologically nonessential metal, but is used as a valuable treatment for mental illness. It is important to study lithium for binding comparisons with other Group 1 metals such as: Na+ is an essential metal for living organisms and has its highest concentration in cells. Sodium is vital in blood pressure and blood coagulation processes. K+ is also a biologically important metal like sodium. Potassium is found in highest concentration inside the cells and acts as a enzyme activator. Additionally it is crucial for plant growth.

What are Amino Acids? Zwitterion Neutral Amino acids are tetrahedral compounds with an amine group attached to the alpha carbon with the general structure form shown below, where R is a side chain that gives the amino acid specific properties. Since amino acids (except glycine) are chiral, both the L and D forms are possible structures. The L form of amino acids, however, are the only forms produced in nature and are vital to cell life. Amino acids form non-covalent interactions and hydrogen bond together forming peptides and proteins that do all cellular work. Typically, the physiological pH range causes the neutral carboxylate to lose a proton while the amine group will pick up the proton resulting in the zwitterion structure. Zwitterion Neutral

20 Amino Acids... There are 20 amino acids that form peptides, which polymerize to make up allmammalian proteins. As shown to the left the amino acids can be separated into four groups based on their R group properties: Non-polar and hydrophobic Polar and hydrophilic Basic Acidic In this study the behavior of the sulfur containing amino acids, methionine and cysteine, is examined.

MetNa+

MetLi+

MetK+

References Wilkins, Patricia C. & Ralph G. Wilkins. Inorganic Chemistry in Biology. Oxford Science Publications. 1997: 2, 10, 29-30, 37. Mathews, Christopher K. & K.E. Van Holde. Biochemistry. Benjamin/Cummings Publishing. 2nd ed. 1996: 129- 136, 744.