The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

The minor groove hydration spine is a key feature of the crystal structure of the B-DNA dodecamer duplex [d(CGCGAATTCGCG)]2. At the floor of the groove, water molecules bridge bases from opposite strands by hydrogen bonding to N3 and O2 atoms of adenine and thymine, respectively. However, the interpretation that the series of electron density peaks lining the groove represents indeed water molecules, while generally agreed upon, remains an assumption. The limited resolutions of dodecamer crystal structures have thus far made it impossible to reliably distinguish between water and monovalent metal cations, such as Na+, normally present in the crystallization buffer. Using X-ray diffraction data to near-atomic resolution of dodecamer crystals grown in the presence of either Rb+ or Cs+ cacodylate, we have tested the possibility of alkali metal ion coordination in the minor groove. The structural data are consistent with a single Rb+ intruding the hydration spine at the central ApT step. The ion has partial occupancy and replaces the water molecule that links the keto oxygens of thymines from opposite strands. The observed dimensions of the binding site suggest preferred binding of Rb+ or K+, while Na+ or Cs+ may be prevented from binding stably. Therefore, minor groove ion coordination appears to be an isolated event, highly sequence dependent and unlikely to significantly affect the particular geometry of the A-tract in the Dickerson−Drew dodecamer. In addition to allowing a distinction between water and alkali metal ions, the high-resolution crystal structures provide a more complete picture of the minor groove water structure:  four fused water hexagons dissect the central portion of the minor groove, with the inner corners of the hexagons coinciding with the original spine water positions. Thus, it may be more appropriate to refer to this arrangement as a ribbon of hydration instead of a spine of hydration.

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a ''hybrid'' droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as Ϸ140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in Ϸ10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 l of protein solution, Ϸ1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.droplets ͉ plugs ͉ protein structure ͉ high-throughput ͉ miniaturization T his work reports a ''hybrid'' microfluidic approach that uses nanoliter plugs to perform screening and optimization simultaneously in the same experiment. To validate this method using a challenging problem, we demonstrate its compatibility with crystallization of membrane proteins. Small-scale screening and optimization experiments are important for biological assays, chemical screening, and protein crystallization (1-3). Screening and optimization are usually carried out sequentially. In the case of protein crystallization, random sparse matrix screening initially identifies the precipitants that may lead to crystallization. Subsequent gradient optimization establishes concentrations of these precipitants that lead to diffractionquality crystals (4). Combining screening and optimization steps into a single hybrid experiment would eliminate the need to wait for the outcome of the initial screen before carrying out subsequent optimizations. Furthermore, a hybrid experiment would reduce the false negatives (5) associated with screens performed at a single concentration. The hybrid experiment could also be more conclusive, because a single batch of the s...

KcsA is a proton-activated, voltage-modulated K ؉ channel that has served as the archetype pore domain in the Kv channel superfamily. Here, we have used synthetic antigen-binding fragments (Fabs) as crystallographic chaperones to determine the structure of full-length KcsA at 3.8 Å, as well as that of its isolated C-terminal domain at 2.6 Å. The structure of the full-length KcsA-Fab complex reveals a well-defined, 4-helix bundle that projects Ϸ70 Å toward the cytoplasm. This bundle promotes a Ϸ15°bending in the inner bundle gate, tightening its diameter and shifting the narrowest point 2 turns of helix below. Functional analysis of the full-length KcsA-Fab complex suggests that the C-terminal bundle remains whole during gating. We suggest that this structure likely represents the physiologically relevant closed conformation of KcsA.

Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.