A large increase in enzyme-substrate affinity by protein engineering

A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme-substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 leads to Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 leads to Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.     

TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function

Progressive kidney failure is a genetically and clinically heterogeneous group of disorders. Podocyte foot processes and the interposed glomerular slit diaphragm are essential components of the permeability barrier in the kidney. Mutations in genes encoding structural proteins of the podocyte lead to the development of proteinuria, resulting in progressive kidney failure and focal segmental glomerulosclerosis. Here, we show that the canonical transient receptor potential 6 (TRPC6) ion channel is expressed in podocytes and is a component of the glomerular slit diaphragm. We identified five families with autosomal dominant focal segmental glomerulosclerosis in which disease segregated with mutations in the gene TRPC6 on chromosome 11q. Two of the TRPC6 mutants had increased current amplitudes. These data show that TRPC6 channel activity at the slit diaphragm is essential for proper regulation of podocyte structure and function.     

Catecholamine content of the arterial walls in experimental hypertension

Zusammenfassung In Hunden mitGoldblatt-Hypertonie ist die Adrenalinkonzentration der Arterien stark erhöht, diejenige von Noradrenalin aber — verglichen mit den Werten der Kontrolltiere — erniedrigt. Die Adrenalinerhöhung in der Arterienwandung ist wahrscheinlich nicht die Ursache des Hochdruckes, da sie auch in Hunden ohne postoperative Blutdruckerhöhung gefunden wurde.     

Biosynthesis of plant sterols. Conversion of cholesterol to pregnenolone inDigitalis purpurea

Résumé Un échantillon purifié de cholestérol-4-C¹⁴ a été absorbé par des feuilles de 2 plantes deDigitalis purpurea. Après 18 jours on a extrait les plantes et séparé les composés radioactifs. Environ 1% des métabolites globales a été constaté en forme de pregnénolone. Ainsi le cholestérol peut agir comme précurseur des stérols végétaux.

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This work was supported by Grants No. CA-07137, No. A-5326 and No. FR-05528 from the U.S. Public Health Service, and No. G-21799 from the National Science Foundation.     

Biosynthesis of plant sterols. Conversion of cholesterol to pregnenolone in Digitalis purpurea

Zusammenfassung Der bei der Bestrahlung mit ionisierenden Strahlen auftretende Sättigungseffekt, d.h. die Abweichung von der Linearität des Verhältnisses zwischen der Strahlendosis und der Zahl der geschädigten Molekeln bei höheren Dosisleistungen, wird unter Zugrundelegung eines einfachen Modells, welches sowohl die Erzeugung als auch die Rekombination der Strahlenschäden berücksichtigt, mathematisch beschrieben.     

TRIM24 links a non-canonical histone signature to breast cancer

Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.     

Making antibody fragments using phage display libraries

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.