Protein stability and structure in HIC: hydrogen exchange experiments and COREX calculations

Hydrogen exchange mass spectrometry (HXMS) coupled to proteolytic digestion has been used to probe the conformation of bovine β-lactoglobulin (BLG), bovine α-lactalbumin (BLA), and human serum albumin (HSA) in solution and while adsorbed to the hydrophobic interaction chromatography media Phenyl Sepharose 6FF. All three proteins show evidence of EX1 exchange kinetics, indicating a loss of stability on the surface. HX protection patterns for all three proteins also indicate that the unfolded form is only partially solvent exposed. The hydrogen-deuterium exchange patterns of BLG and BLA on the surface suggest a structure that resembles each protein's respective solution phase molten globule state. The low stability of Domain II of HSA observed on Phenyl Sepharose 6FF also suggests a link to solution stability because Domain II is frequently cited as the least stable domain in solution unfolding pathways. COREX, an algorithm used to compute protein folding stabilities, correctly predicts solution hydrogen-deuterium exchange patterns for BLG and offers insight into its adsorbed phase stabilities but is unreliable for BLA predictions. The results of this work demonstrate a link between solution-phase local stability patterns and the nature of partially unfolded states that proteins can adopt on HIC surfaces.     

Origins of enhanced proton transport in the Y7F mutant of human carbonic anhydrase II

Human carbonic anhydrase II (HCA II), among the fastest enzymes known, catalyzes the reversible hydration of CO 2 to HCO 3 (-). The rate-limiting step of this reaction is believed to be the formation of an intramolecular water wire and transfer of a proton across the active site cavity from a zinc-bound solvent to a proton shuttling residue (His64). X-ray crystallographic studies have shown this intramolecular water wire to be directly stabilized through hydrogen bonds via a small well-defined set of amino acids, namely, Tyr7, Asn62, Asn67, Thr199, and Thr200. Furthermore, X-ray crystallographic and kinetic studies have shown that the mutation of tyrosine 7 to phenylalanine, Y7F HCA II, has the effect of increasing the proton transfer rate by 7-fold in the dehydration direction of the enzyme reaction compared to wild-type (WT). This increase in the proton transfer rate is postulated to be linked to the formation of a more directional, less branched, water wire. To evaluate this proposal, molecular dynamics simulations have been employed to study water wire formation in both the WT and Y7F HCA II mutant. These studies reveal that the Y7F mutant enhances the probability of forming small water wires and significantly extends the water wire lifetime, which may account for the elevated proton transfer seen in the Y7F mutant. Correlation analysis of the enzyme and intramolecular water wire indicates that the Y7F mutant significantly alters the interaction of the active site waters with the enzyme while occupancy data of the water oxygens reveals that the Y7F mutant stabilizes the intramolecular water wire in a manner that maximizes smaller water wire formation. This increase in the number of smaller water wires is likely to elevate the catalytic turnover of an already very efficient enzyme.     

3d-Metal derivatives of the [Cu(I)(SO3)4]7- ion: structure and magnetism

The Cu(SO(3))(4)(7-) anion, which consists of a tetrahedrally coordinated Cu(I) centre coordinated to four sulfur atoms, is able to act as a multidentate ligand in discrete and infinite supramolecular species. The slow oxidation of an aqueous solution of Na(7)Cu(SO(3))(4) yields a mixed oxidation state, 2D network of composition Na(5){[Cu(II)(H(2)O)][Cu(I)(SO(3))(4)]}·6H(2)O. The addition of Cu(II) and 2,2'-bipyridine to an aqueous Na(7)Cu(SO(3))(4) solution leads to the formation of a pentanuclear complex of composition {[Cu(II)(H(2)O)(bipy)](4)[Cu(I)(SO(3))(4)]}(+); a combination of hydrogen bonding and π-π stacking interactions leads to the generation of infinite parallel channels that are occupied by disordered nitrate anions and water molecules. A pair of Cu(SO(3))(4)(7-) anions each act as a tridentate ligand towards a single Mn(II) centre when Mn(II) ions are combined with an excess of Cu(SO(3))(4)(7-). An anionic pentanuclear complex of composition {[Cu(I)(SO(3))(4)](2)[Fe(III)(H(2)O)](3)(O)} is formed when Fe(II) is added to a Cu(+)/SO(3)(2-) solution. Hydrated ferrous [Fe(H(2)O)(6)(2+)] and sodium ions act as counterions for the complexes and are responsible for the formation of an extensive hydrogen bond network within the crystal. Magnetic susceptibility studies over the temperature range 2-300 K show that weak ferromagnetic coupling occurs within the Cu(II) containing chains of Na(5){[Cu(II)(H(2)O)][Cu(I)(SO(3))(4)]}·6H(2)O, while zero coupling exists in the pentanuclear cluster {[Cu(II)(H(2)O)(bipy)](4)[Cu(I)(SO(3))(4)]}(NO(3))·H(2)O. Weak Mn(II)-O-S-O-Mn(II) antiferromagnetic coupling occurs in Na(H(2)O)(6){[Cu(I)(SO(3))(4)][Mn(II)(H(2)O)(2)](3)}, the latter formed when Mn was in excess during synthesis. The compound, Na(3)(H(2)O)(6)[Fe(II)(H(2)O)(6)](2){[Cu(I)(SO(3))(4)](2)[Fe(III)(H(2)O)](3)(O)}·H(2)O, contained trace magnetic impurities that affected the expected magnetic behaviour.