Ann E. McDermott

Structure and Dynamics of Membrane Proteins by Magic Angle Spinning Solid-State NMR

Membrane proteins remain difficult to study by traditional methods. Magic angle spinning solid-state NMR (MAS SSNMR) methods present an important approach for studying membrane proteins of moderate size. Emerging MAS SSNMR methods are based on extensive assignments of the nuclei as a basis for structure determination and characterization of function. These methods have already been used to characterize fibrils and globular proteins and are being increasingly used to study membrane proteins embedded in lipids. This review highlights recent applications to intrinsic membrane proteins and summarizes recent technical advances that will enable these methods to be utilized for more complex membrane protein systems in the near future.

Optimal alignment for enzymatic proton transfer: structure of the Michaelis complex of triosephosphate isomerase at 1.2-A resolution.

In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the α-hydroxy carbons of the substrate (C… O ≈ 3.0 Å), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrates O1 and O2 (N… O ≤ 3.0 and 2.6 Å, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The ɛ ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrates ketone oxygen, the bridging oxygen, and a terminal phosphates oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.

The state of manganese in the photosynthetic apparatus: 4. Structure of the manganese complex in Photosystem II studied using EXAFS spectroscopy. The S1 state of the O2-evolving Photosystem II complex from spinach

Abstract The structure of the Mn complex in the oxygen-evolving system and its mechanistic relation to photosynthetic oxygen evolution are poorly understood, though many studies have established that membrane-bound Mn plays an active role. Recently established procedures for isolating oxygen-evolving subchloroplast Photosystem II (PS II) preparations and the discovery of a light-induced multiline EPR signal attributable to the S2 state of the O2-evolving complex have facilitated the preparation of samples well characterized in the S1 and S2 states. We have used extended X-ray absorption fine structure (EXAFS) spectroscopy to probe the ligand environment of Mn in PS II particles from spinach, and in this report we present our results. The essential feature of the EXAFS results are that at least two Mn atoms per PS II reaction center occur as a binuclear species with a metal-metal distance of approx. 2.7 A, with low Z atoms, N or O, at a distance of approx. 1.75 A and at approx. 1.98 A, which are characteristic of bridging and terminal ligands. These results agree well with those derived from whole chloroplasts that provided the first evidence for a binuclear manganese complex (Kirby, J.A., Robertson, A.S., Smith, J.P., Thompson, A.C., Cooper, S.R. and Klein, M.P. (1981) J. Am. Chem. Soc. 103, 5529–5537).

Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage

This study has examined the atomic-level dynamics of the protein in the capsid of filamentous phage Pf1. This capsid consists of ≈7,300 small subunits of only 46 aa in a helical array around a highly extended, circular single-stranded DNA molecule of 7,349 nt. Measurements were made of site-specific, solid-state NMR order parameters, 〈S〉, the values which are dimensionless quantities between 0 (mobile) and 1 (static) that characterize the amplitudes of molecular bond angular motions that are faster than microseconds. It was found that the protein subunit backbone is very static, and of particular interest, it appears to be static at residues glycine 15 and glutamine 16 where it had been previously thought to be mobile. In contrast to the backbone, several side chains display large-amplitude angular motions. Side chains on the virion exterior that interact with solvent are highly mobile, but surprisingly, the side chains of residues arginine 44 and lysine 45 near the DNA deep in the interior of the virion are also highly mobile. The large-amplitude dynamic motion of these positively charged side chains in their interactions with the DNA were not previously expected. The results reveal a highly dynamic aspect of a DNA–protein interface within a virus.

Conformational Dynamics in the Selectivity Filter of KcsA in Response to Potassium Ion Concentration

Conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid-state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the potassium ion concentration is changed from 50 mM to 1 muM. Potassium ion titration studies reveal that the site-specific K(d) for K(+) binding at the key pore residue Val76 is on the order of approximately 7 muM and that a relatively high sample hydration is necessary to observe the low-K(+) conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high-K(+) and low-K(+) states are in slow exchange on the NMR timescale (k(ex)<500 s(-)(1)). The slow rate and tight binding for evacuating both inner sites simultaneously differ from prior observations in detergent in solution, but agree well with measurements by electrophysiology and appear to result from our use of a hydrated bilayer environment. These observations strongly support a common assumption that the low-K(+) state is not involved in ion transmission, and that during transmission one of the two inner sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of the evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.

Assignment of the g = 4.1 ERP signal to manganese in the S2 state of the photosynthetic oxygen-evolving complex: An X-ray absorption edge spectroscopy study☆

X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O2-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S2 multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S2 state.

Protonation state of E71 in KcsA and its role for channel collapse and inactivation

The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solid-state NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K+ titrations, we detect a local change at E71 that is coupled to channel collapse at low K+. To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its hydrogen bond partner D80, and use the tensors to assign protonation states to E71 and D80 at high K+ and neutral pH. Our measurements show that E71 is protonated at pH 7.5 and must have an unusually perturbed pKa (> 7.5) suggesting that the change at E71 is a structural rearrangement rather than a protonation event. The results offer new mechanistic insights into why the widely used mutant KcsA–E71A does not inactivate and establish the ambient K+ level as a means to populate the inactivated state of KcsA in a controlled way.

Hydrogen bonding effects on amine rotation rates in crystalline amino acids

Rates of rotation for amines in a variety of crystalline environments are reported, and the trends are explained in terms of the strengths of local hydrogen bonding interactions. Proton spin-lattice relaxation times (T1) and deuterium broad-line NMR spectra have been measured for D-, DL- and L- aspartic acid, two polymorphs of glycine, alanine, and leucine in the temperature range from -40 to 110 degrees C. The energy barriers for amine rotation are 27 +/- 2 kJ mol-1 for D- or L-aspartic acid and 22 +/- 2 kJ mol-1 for DL-aspartic acid; these energies are slightly lower than the previously reported value for the L from based on direct proton T1 measurements at 60 MHz. The values for the alpha and gamma forms of glycine were 24 +/- 2 and 30 +/- 2 kJ mol-1 respectively, that for L-alanine was 40 +/- 2 and that for L-leucine was 49 +/- 3 kJ mol-1. These are all in rough agreement with previously reported values (although the differences for the polymorphs of glycine and for L- vs. DL-aspartic acid were not reported). Crystal structures of these amino acids indicate differences in hydrogen bonding environments around the R-NH3+ groups that are probably responsible for the different activation barriers. A molecular mechanics calculation of the rotation energy barriers for L- and DL-aspartic acid based on the crystal structures gave satisfactory agreement with experimental results if a uniform (and arbitrarily chosen) dielectric constant of 2.5 was assumed. Differences between L- and DL-aspartic acids and between two polymorphs of glycine were well represented qualitatively. Including additional neighboring molecules not involved in the hydrogen bonding or including periodic boundary conditions to describe the crystal packing did not significantly affect these results. If vacuum dielectric constants are used, the barriers are uniformly overestimated, and if the experimental macroscopic dielectric constant values are used, the barriers are generally underestimated. Dielectric constants differ substantially from one amino acid to another and significantly affect the estimated barriers; in fact, the bulk dielectric constants appear to be the major difference between the highest and the lowest values. The difficulty of accurately including dielectric relaxation into molecular mechanics calculations resulted in the disagreement between experimental measurements and theoretical calculations.

Transmembrane allosteric coupling of the gates in a potassium channel

Significance K+ channels are spontaneously inactivated subsequent to channel opening, a process that is central to their many crucial roles in cell signaling. Several studies suggest that for KcsA inactivation involves ion release. Here we demonstrate that K+ expulsion from the selectivity filter at neutral pH causes spontaneous opening. This illustrates an unexpected opening mechanism for KcsA, which is normally pH gated, and also provides strong evidence for transmembrane allosteric coupling. It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.

Protein–ice interaction of an antifreeze protein observed with solid-state NMR

NMR on frozen solutions is an ideal method to study fundamental questions of macromolecular hydration, because the hydration shell of many biomolecules does not freeze together with bulk solvent. In the present study, we present previously undescribed NMR methods to study the interactions of proteins with their hydration shell and the ice lattice in frozen solution. We applied these methods to compare solvent interaction of an ice-binding type III antifreeze protein (AFP III) and ubiquitin a non-ice-binding protein in frozen solution. We measured 1H-1H cross-saturation and cross-relaxation to provide evidence for a molecular contact surface between ice and AFP III at moderate freezing temperatures of -35 °C. This phenomenon is potentially unique for AFPs because ubiquitin shows no such cross relaxation or cross saturation with ice. On the other hand, we detected liquid hydration water and strong water–AFP III and water–ubiquitin cross peaks in frozen solution using relaxation filtered 2H and HETCOR spectra with additional 1H-1H mixing. These results are consistent with the idea that ubiquitin is surrounded by a hydration shell, which separates it from the bulk ice. For AFP III, the water cross peaks indicate that only a portion of its hydration shell (i.e., at the ice-binding surface) is in contact with the ice lattice. The rest of AFP III’s hydration shell behaves similarly to the hydration shell of non-ice-interacting proteins such as ubiquitin and does not freeze together with the bulk water.