Echo-delay resolution in sonar images of the big brown bat, Eptesicus fuscus

Echolocating big brown bats (Eptesicus fuscus) broadcast ultrasonic frequency-modulated (FM) biosonar sounds (20–100 kHz frequencies; 10–50 μs periods) and perceive target range from echo delay. Knowing the acuity for delay resolution is essential to understand how bats process echoes because they perceive target shape and texture from the delay separation of multiple reflections. Bats can separately perceive the delays of two concurrent electronically generated echoes arriving as little as 2 μs apart, thus resolving reflecting points as close together as 0.3 mm in range (two-point threshold). This two-point resolution is roughly five times smaller than the shortest periods in the bat’s sounds. Because the bat’s broadcasts are 2,000–4,500 μs long, the echoes themselves overlap and interfere with each other, to merge together into a single sound whose spectrum is shaped by their mutual interference depending on the size of the time separation. To separately perceive the delays of overlapping echoes, the bat has to recover information about their very small delay separation that was transferred into the spectrum when the two echoes interfered with each other, thus explicitly reconstructing the range profile of targets from the echo spectrum. However, the bat’s 2-μs resolution limit is so short that the available spectral cues are extremely limited. Resolution of delay seems overly sharp just for interception of flying insects, which suggests that the bat’s biosonar images are of higher quality to suit a wider variety of orientation tasks, and that biosonar echo processing is correspondingly more sophisticated than has been suspected.

Structural features of the γ subunit of the Escherichia coli F1 ATPase revealed by a 4.4-Å resolution map obtained by x-ray crystallography

The F1 part of the F1FO ATP synthase from Escherichia coli has been crystallized and its structure determined to 4.4-Å resolution by using molecular replacement based on the structure of the beef-heart mitochondrial enzyme. The bacterial F1 consists of five subunits with stoichiometry α3, β3, γ, δ, and ɛ. δ was removed before crystallization. In agreement with the structure of the beef-heart mitochondrial enzyme, although not that from rat liver, the present study suggests that the α and β subunits are arranged in a hexagonal barrel but depart from exact 3-fold symmetry. In the structures of both beef heart and rat-liver mitochondrial F1, less than half of the structure of the γ subunit was seen because of presumed disorder in the crystals. The present electron-density map includes a number of rod-shaped features which appear to correspond to additional α-helical regions within the γ subunit. These suggest that the γ subunit traverses the full length of the stalk that links the F1 and FO parts and makes significant contacts with the c subunit ring of FO.

Biosynthesis of vitamin B12: Concerning the identity of the two-carbon fragment eliminated during anaerobic formation of cobyrinic acid

It has been proved that, during anaerobic biosynthesis of the corrin macrocycle, the two-carbon fragment excised from the precursor, precorrin-3, is acetaldehyde, which originates from C-20 and its attached methyl group. This apparently contradictory finding is rationalized in terms of the subsequent enzymatic oxidation of acetaldehyde to acetic acid, which was previously regarded as the volatile fragment released by the action of the biosynthetic enzymes of Propionibacterium shermanii. The observation that acetaldehyde (rather than acetic acid) is extruded during anaerobic B12 synthesis is in full accord with the structure of factor IV, a new intermediate on the pathway.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the α-band of heme b595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b595 within a few ps, pointing to a direct interaction between hemes b595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

The nature of the excited state of the reaction center of photosystem II of green plants: A high-resolution fluorescence spectroscopy study

We studied the electronically excited state of the isolated reaction center of photosystem II with high-resolution fluorescence spectroscopy at 5 K and compared the obtained spectral features with those obtained earlier for the primary electron donor. The results show that there is a striking resemblance between the emitting and charge-separating states in the photosystem II reaction center, such as a very similar shape of the phonon wing with characteristic features at 19 and 80 cm−1, almost identical frequencies of a number of vibrational modes, a very similar double-Gaussian shape of the inhomogeneous distribution function, and relatively strong electron-phonon coupling for both states. We suggest that the emission at 5 K originates either from an exciton state delocalized over the inactive branch of the photosystem or from a fraction of the primary electron donor that is long-lived at 5 K. The latter possibility can be explained by a distribution of the free energy difference of the primary charge separation reaction around zero. Both possibilities are in line with the idea that the state that drives primary charge separation in the reaction center of photosystem II is a collective state, with contributions from all chlorophyll molecules in the central part of the complex.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at ≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 × 3.2 × 1.2 μm3) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455–476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, ≈66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.

High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex.

We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATA-binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFIIA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes.

Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

Mapping the protein regions responsible for the functional specificities of the Arabidopsis MADS domain organ-identity proteins.

The Arabidopsis MADS domain proteins AP1, AP3, PI, and AG specify floral organ identity. All of these proteins contain a MADS domain required for DNA binding and dimerization; a region termed L (linker between MADS domain and K domain), which plays an important role in dimerization specificity; the K domain, named for its similarity to the coiled-coil domain of keratin; and a C-terminal region of unknown function. To determine which regions of these proteins are responsible for their abilities to specify different organs, we have made a number of chimeric MADS box genes. The in vivo function of these chimeric genes was investigated by ectopic expression in transgenic Arabidopsis plants. The four proteins fall into two classes on the basis of regions responsible for their functional specificities. The L region and K domain define the functional specificities of AP3 and PI, while the MADS domain and L region define the functional specificities of AP1 and AG.

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

Beta-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detailed structural and mechanistic understanding of these enzymes can be expected to guide the design of new antibacterial compounds resistant to their action. A number of high-resolution structures are available for class A beta-lactamases, whose catalytic mechanism involves the acylation of a serine residue at the active site. The identity of the general base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine residue and a glutamic acid residue having been proposed as candidates for this role. We have used the pH dependence of chemical modification of epsilon-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the epsilon-methylene 1H and 13C chemical shifts (in enzyme selectively labeled with [epsilon-13C]lysine) to estimate the pKa of the relevant lysine residue, lysine-73, of TEM-1 beta-lactamase. Both methods show that the pKa of this residue is > 10, making it very unlikely that this residue could act as a proton acceptor in catalysis. An alternative mechanism in which this role is performed by glutamate-166 through an intervening water molecule is described.

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.