Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface

Kouadio JL, Horn JR, Pal G, Kossiakoff AA

J. Biol. Chem. 2005 Jul;280(27):25524-32

PMID: 15857837


The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we …

Continue reading

Total chemical synthesis and X-ray crystal structure of a protein diastereomer: [D-Gln 35]ubiquitin

Bang D, Makhatadze GI, Tereshko V, Kossiakoff AA, Kent SB

Angew. Chem. Int. Ed. Engl. 2005 Jun;44(25):3852-6

PMID: 15834850


Intramolecular cooperativity in a protein binding site assessed by combinatorial shotgun scanning mutagenesis

Pál G, Ultsch MH, Clark KP, Currell B, Kossiakoff AA, Sidhu SS

J. Mol. Biol. 2005 Apr;347(3):489-94

PMID: 15755445


Combinatorial shotgun alanine-scanning was used to assess intramolecular cooperativity in the high affinity site (site 1) of human growth hormone (hGH) for binding to its receptor. A total of 19 side-chains were analyzed and statistically …

Continue reading

The crystal structure of a quercetin 2,3-dioxygenase from Bacillus subtilis suggests modulation of enzyme activity by a change in the metal ion at the active site(s)

Gopal B, Madan LL, Betz SF, Kossiakoff AA

Biochemistry 2005 Jan;44(1):193-201

PMID: 15628860


Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with …

Continue reading

The high- and low-affinity receptor binding sites of growth hormone are allosterically coupled

Walsh ST, Sylvester JE, Kossiakoff AA

Proc. Natl. Acad. Sci. U.S.A. 2004 Dec;101(49):17078-83

PMID: 15563602


Growth hormone regulates its biological properties via a sequential hormone-induced receptor homodimerization mechanism. Using a mutagenesis-scanning analysis of 81 single and 32 pairwise double mutations, we show that the hormone’s two spatially distal receptor binding sites (Site1 and Site2) …

Continue reading

The structural basis for biological signaling, regulation, and specificity in the growth hormone-prolactin system of hormones and receptors

Kossiakoff AA

Adv. Protein Chem. 2004;68:147-69

PMID: 15500861


The pituitary hormones growth hormone (GH), prolactin (PRL) and placental lactogen (PL), are members of an extensive cytokine superfamily of hormones and receptors that share many of the same general structure-function relationships in expressing their biological activities. The biology of the pituitary hormones involves a very …

Continue reading

Dissecting the binding energy epitope of a high-affinity variant of human growth hormone: cooperative and additive effects from combining mutations from independently selected phage display mutagenesis libraries

Bernat B, Sun M, Dwyer M, Feldkamp M, Kossiakoff AA

Biochemistry 2004 May;43(20):6076-84

PMID: 15147191


Phage display mutagenesis is a widely used approach to engineering novel protein properties and is especially powerful in probing structure-function relationships in molecular recognition processes. The relative contributions of additive and cooperative binding forces and the influence of conformational …

Continue reading

Site2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization

Walsh ST, Jevitts LM, Sylvester JE, Kossiakoff AA

Protein Sci. 2003 Sep;12(9):1960-70

PMID: 12930995


Receptor signaling in the growth hormone (GH)-growth hormone receptor (GHR) system is controlled through a sequential two-step hormone-induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of …

Continue reading

The functional binding epitope of a high affinity variant of human growth hormone mapped by shotgun alanine-scanning mutagenesis: insights into the mechanisms responsible for improved affinity

Pal G, Kossiakoff AA, Sidhu SS

J. Mol. Biol. 2003 Sep;332(1):195-204

PMID: 12946357


A high-affinity variant of human growth hormone (hGH(v)) contains 15 mutations within site 1 and binds to the hGH receptor (hGHR) approximately 400-fold tighter than does wild-type (wt) hGH (hGH(wt)). We used shotgun scanning combinatorial mutagenesis to dissect the energetic contributions …

Continue reading

The first semi-synthetic serine protease made by native chemical ligation

Pál G, Santamaria F, Kossiakoff AA, Lu W

Protein Expr. Purif. 2003 Jun;29(2):185-92

PMID: 12767808


Selective incorporation of non-natural amino acid residues into proteins is a powerful approach to delineate structure-function relationships. Although many methodologies are available for chemistry-based protein engineering, more facile methods are needed to make this approach suitable for routine laboratory …

Continue reading