John B. C. Findlay

Met-204 and Tyr-205 are together important for binding GLP-1 receptor agonists but not their N-terminally truncated analogues.

A mutagenesis study to systematically analyse residues spanning the first extracellular loop of the GLP-1 receptor identified a double mutant, Met-204/Tyr-205-Ala/Ala, which displayed: markedly reduced affinity for the natural agonist GLP-1; slightly reduced affinity for its analogue exendin-4; and unaltered affinity for several N-terminally truncated analogues of GLP-1 and exendin-4. This suggests that the locus is important for the formation of the binding site for the N-terminal residues of peptide agonists.

The structure of β-lactoglobulin and its similarity to plasma retinol-binding protein

Since its first isolation1, bovine β-lactoglobulin (BLG) has been an enigma: although it is abundant in the whey fraction of milk, its function is still not clear. The results of the many physicochemical studies on the protein need a structural interpretation. We report here the structure of the orthorhombic crystal form of cow BLG at pH 7.6, at a resolution of 2.8 Å. It has an unusual protein fold, composed of two slabs of antiparallel β-sheet, which shows a remarkable similarity to plasma retinol-binding protein. A possible binding site for retinol in BLG has been identified by model-building. This suggests a role for BLG in vitamin A transport and we have discovered specific receptors for the BLG–retinol complex in the intestine of neonate calves.

PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules

SummaryA software package is described that operates on small molecules observed in the PDB collection of protein structures. Molecular topology files for many molecular modeling programs can be generated automatically. The three-dimensional coordinates of small molecules can be converted to molecular descriptor strings that encode them uniquely in order to enable small-molecule recognition, despite high variability in atom and molecule nomenclature. From this descriptor a plausible 3D structure can be regenerated using energy minimisation. Alternatively, an ensemble of structures can be generated using a distance-geometry-based algorithm.

Three-dimensional modelling of G protein-linked receptors

Members of the G protein-linked receptor superfamily have not yet yielded to X-ray crystallography. However, diffraction data from other membrane-bound receptors - the photosynthetic reaction centre and bacteriorhodopsin - have provided some information that may also apply to the G protein family. John Findlay and Elias Eliopoulos integrate this information together with analysis of amino acid sequences from cloned receptors, to derive workable three-dimensional models of these proteins. Such models identify ligand-binding and G protein-associating domains.

MAST/Orbit has a role in microtubule–kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.

A comparison of techniques for calculating protein essential dynamics

Recently the basic theory of essential dynamics, a method for extracting large concerted motions from protein molecular dynamics trajectories, was described. Here, we introduce and test new aspects. A method for diagonalizing large covariance matrices is presented. We show that it is possible to perform essential dynamics using different subsets of atoms and compare these to the basic C‐α analysis. Essential dynamics analyses are also compared to the normal modes method. The stability of the essential space during a simulation is investigated by comparing the two halves of a trajectory. Apart from the analyses in Cartesian space, the essential dynamics in ϕ/ψ torsion angle space is discussed.

A novel mutation within the rhodopsin gene (Thr‐94‐Ile) causing autosomal dominant congenital stationary night blindness

More than 100 mutations within the rhodopsin gene have been found to be responsible for some forms of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and subsequent disturbance of day vision that may eventually result in total blindness. Congenital stationary night blindness (CSNB) is an uncommon inherited retinal dysfunction in which patients complain of night vision difficulties of a nonprogressive nature only and in which generally there is no involvement of day vision. We report the results of molecular genetic analysis of an Irish family segregating an autosomal dominant form of CSNB in which a previously unreported threonine‐to‐isoleucine substitution at codon 94 in the rhodopsin gene was found to segregate with the disease. Computer modeling suggests that constitutive activation of transducin by the altered rhodopsin protein may be a mechanism for disease causation in this family. Only two mutations within the rhodopsin gene have been previously reported in patients with congenital stationary night blindness, constitutive activation also having been proposed as a possible disease mechanism. Hum Mutat 13:75–81, 1999.

The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP" reductases

Three soluble ferrisiderophore reductases (FsrA, FsrB and FsrC) were detected in Escherichia coli. FsrB was purified and identified as the haemoglobin‐like protein (HMP) by size and N‐terminal sequence analyses. HMP was previously isolated as a dihydropteridine reductase and is now shown to have ferrisiderophore reductase activity. Database searches revealed that the C‐terminal region of HMP (FsrB) is homologous to members of a family of flavoprotein oxidoreductases which includes ferredoxin NADP+ reductase (FNR). The combination of FNR‐like and haemoglobin‐like regions in HMP (FsrB) represents a novel pairing of functionally and structurally distinct domains. Structure—function properties of other FNR‐like proteins, including LuxG and VanB, are also discussed.

Ligand binding characteristics of homologous rat and mouse urinary proteins and pyrazine-binding protein of calf

1. The binding affinities of three classes of homologous proteins, alpha 2u protein from rat urine, major urinary protein of mouse and pyrazine-binding protein of calf nasal mucosa have been determined for a panel of ligands. 2. Best ligands were low threshold odorants for man, but chemically unrelated. 3. The binding spectra of the homologous proteins were different.

Homology between the pyrazine-binding protein from nasal mucosa and major urinary proteins.

Sequence analysis of the pyrazine‐binding protein from bovine olfactory mucosa reveals marked homology with a family of proteins of unknown function found in the urine of the adult male mouse and rat. In view of the dramatic biological responses to odorants transmitted in male rodent urines, it is proposed that these proteins play important roles in some aspects of odor transmission and reception.