Structure of a copper-mediated base pair in DNA.

Stable and selective DNA base pairing by metal coordination was recently demonstrated with nucleotides containing complementary pyridine-2,6-dicarboxylate (Dipic) and pyridine (Py) bases (Meggers, E.; Holland, P. L.; Tolman; W. B.; Romesberg, F. E.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 10714-10715). To understand the structural consequences of introducing this novel base pair into DNA we have solved the crystal structure of a duplex containing the metallo-base pair. The structure shows that the bases pair as designed, but in a Z-DNA conformation. The structure also provides a structural explanation for the B- to Z-DNA transition in this duplex. Further solution studies demonstrate that the metallo-base pair is compatible with Z- or B-DNA conformations, depending on the duplex sequence.

Selection for improved subtiligases by phage display.

Engineering enzyme activity has been challenging because of uncertainties in structure-function relationships and difficulties in screening a large number of mutant enzymes. A product capture strategy using phage display is presented here for the selection of improved enzymes from a large library of variants (>10(9) independently derived mutants). Subtiligase, a double mutant of subtilisin BPN' that catalyzes the ligation of peptides, was displayed on phage. Twenty-five active site residues were randomly mutated in groups of four or five to yield six different libraries that were independently sorted. Variants that ligated a biotin peptide onto their own extended N termini were selectively captured. Mutant subtiligases were identified that had increased ligase activity. The selection also yielded unexpected subtiligase mutants having residues known to improve the stability and oxidative resistance of wild-type subtilisin. These studies are exemplary for the use of phage to improve enzyme function when it is closely linked to a selectable catalytic event.

Structural plasticity in a remodeled protein-protein interface.

Remodeling of the interface between human growth hormone (hGH) and the extracellular domain of its receptor was studied by deleting a critical tryptophan residue (at position 104) in the receptor, creating a large cavity, and selecting a pentamutant of hGH by phage display that fills the cavity and largely restores binding affinity. A 2.1 A resolution x-ray structure of the mutant complex showed that the receptor cavity was filled by selected hydrophobic mutations of hGH. Large structural rearrangements occurred in the interface at sites that were distant from the mutations. Such plasticity may be a means for protein-protein interfaces to adapt to mutations as they coevolve.

Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library.

Structure-guided phage display was used to select for combinations of interface residues for antibody C(H)3 domains that promote the formation of stable heterodimers. A C(H)3 "knob" mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W. A library of C(H)3 "hole" mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner C(H)3 domain. The C(H)3 knob mutant was fused to a peptide flag and the C(H)3 hole library was fused to M13 gene III. Phage displaying stable C(H)3 heterodimers were recovered by panning using an anti-flag antibody. Phage-selected C(H)3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407'A, and most clones tested were more stable to guanidine hydrochloride denaturation. The thermal stability of individual C(H)3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry. One heterodimer, T366W-T366'S:L368'A:Y407'V, had a t(m) of 69.4 degrees C, which is 4.0 deg.C higher than that for the designed heterodimer and 11.0 deg.C lower than that for the wild-type homodimer. The phage-selected C(H)3 mutant maintained the preference for forming heterodimers over homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay. Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering homodimers for heterodimerization.

Preparing health care organizations for successful case management programs.

This article reports the results of a study of four hospital-based providers in varying stages of implementing case management programs. Three of the providers had most of the necessary elements in place to ensure success, such as a mix of reimbursement sources, an effective and integrated information management system, a full range of clinical services, and continuous quality improvement programs. The authors make several suggestions for key activities that must be pursued by any health care organization seeking to implement a case management program in an era of managed care, tightening reimbursement, and consumer demand for quality care. These include the need to (a) organize essential case management functions under a centralized structure; (b) set realistic, quantifiable targets, and (c) design a communications plan for the program.

Structure of the regulatory domains of the Src-family tyrosine kinase Lck.

The kinase p56lck (Lck) is a T-lymphocyte-specific member of the Src family of non-receptor tyrosine kinases. Members of the Src family each contain unique amino-terminal regions, followed by Src-homology domains SH3 and SH2, and a tyrosine kinase domain. SH3 and SH2 domains mediate critical protein interactions in many signal-transducing pathways. They are small, independently folded modules of about 60 and 100 residues, respectively, and they are often but not always found together in the same molecule. Like all nine Src-family kinases (reviewed in ref. 3), Lck is regulated by phosphorylation of a tyrosine in the short C-terminal tail of its catalytic domain. There is evidence that binding of the phosphorylated tail to the SH2 domain inhibits catalytic activity of the kinase domain and that the SH3 and SH2 domains may act together to effect this regulation. Here we report the crystal structures for a fragment of Lck bearing its SH3 and SH2 domains, alone and in complex with a phosphotyrosyl peptide containing the sequence of the Lck C-terminal regulatory tail. The latter complex represents the regulatory apparatus of Lck. The SH3-SH2 fragment forms similar dimers in both crystals, and the tail peptide binds at the intermolecular SH3/SH2 contact. The two structures show how an SH3 domain might recognize a specific target and suggest how dimerization could play a role in regulating Src-family kinases.

Adolescent life changes and illness: A comparison of three models.

This article examines how stressful life-events relate to the occurrence of illness in a nonclinical random sample of adolescents and what processes intervene in this relationship. Three alternative models of the life events and illness relationship are compared. Data support a "direct stress and illness model" as opposed to either a "sick role behavior model" or a "mental health model." Social support from parents appears to mediate the life events-illness relationship. Undesirable and ambiguous life events have more impact on illness than desirable events do. Results are discussed in terms of adolescence as a unique developmental stage.

Development and evaluation of method for simultaneous determination of phenobarbital and diphenylhydantoin in plasma by high-pressure liquid chromatography.

A procedure for the simultaneous analysis of diphenylhydantoin and phenobarbital in plasma by high-pressure liquid chromatography was developed and evaluated. Separation is effected on a porous particle silicic acid column with chloroform dioxane-isopropanol-acetic acid (310:9.7:1.0:0.1 by volume) and is monitored at 254 nm. Results of the procedure were compared with results of a GLC assay.

Quantitative extraction of tubocurarine, gallamine, and decamethonium from biological materials.

A procedure is described for quantitative extraction of tubocurarine, gallamine, and decamethonium from blood, urine, and tissue. Ion pairs of the drugs are formed with picric acid and are extracted into a dichloromethane/1-pentanol solvent. Qualitative analysis of the extract is effected using thin-layer chromatography. Quantitative analysis is accomplished by elution of TLC areas containing the drug, formation of the ion pair with sodium 9,10-dimethoxyanthracene-2-sulfonate, and analysis of the ion pair by spectrophotofluorometry.