Computational stabilization of human growth hormone

Abstract

Recombinant human growth hormone (hGH) is used worldwide for the treatment of pediatric hypopituitary dwarfism and in children suffering from low levels of hGH. It has limited stability in solution, and because of poor oral absorption, is administered by injection, typically several times a week. Development has therefore focused on more stable or sustained-release formulations and alternatives to injectable delivery that would increase bioavailability and make it easier for patients to use. We redesigned hGH computationally to improve its thermostability. A more stable variant of hGH could have improved pharmacokinetics or enhanced shelf-life, or be more amenable to use in alternate delivery systems and formulations. The computational design was performed using a previously developed combinatorial optimization algorithm based on the dead-end elimination theorem. The algorithm uses an empirical free energy function for scoring designed sequences. This function was augmented with a term that accounts for the loss of backbone and side-chain conformational entropy. The weighting factors for this term, the electrostatic interaction term, and the polar hydrogen burial term were optimized by minimizing the number of mutations designed by the algorithm relative to wild-type. Forty-five residues in the core of the protein were selected for optimization with the modified potential function. The proteins designed using the developed scoring function contained six to 10 mutations, showed enhancement in the melting temperature of up to 16°C, and were biologically active in cell proliferation studies. These results show the utility of our free energy function in automated protein design.

A thermodynamic study on the interaction between magnesium ion and human growth hormone

“…The molecular weight of hGH was taken to be 22 kDa. 4,6 Temperature-Scanning Spectroscopy. Absorbance profiles, which describe the thermal denaturation of hGH, were obtained from a UV-visible spectrophotometer CARY-100-Bio model fitted with a temperature programmer, which controls the speed of temperature change in melting experiments.…”

“…The molecular mass is approximately 22 kDa, with pI 5.3. [2][3][4][5][6] There are two disulfide bridges present in the protein: one connecting distant parts of the molecule involving residues 53 and 165 (large loop) and another near the C terminal between residues 182 and 189 (small loop). 7 Approximately 55% of the polypeptide backbone exists in a right-handed -helical conformation.…”

“…7 Approximately 55% of the polypeptide backbone exists in a right-handed -helical conformation. 6,8 There are some reports on the binding properties and structural changes of hGH due to its interaction with metal ions. [9][10][11][12] The metal-binding site in hGH is located in the hydrophobic core.…”

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

“…Unlike improving substrate-binding or enzyme kinetic properties, protein stability is a function of many variables, from protein folding, core packing, surface electrostatics, to overall rigidity and it appears that these determinants have varying importance in different proteins (Filikov et al, 2002;Permyakov et al, 2005;Strickler et al, 2006). To address these uncertainties computational tools have been developed that can assist with rational thermostability design, and while some of these methods are informative they do not suggest a generalizable strategy that will work for all proteins (Filikov et al, 2002;Potapov et al, 2009). Another successful approach is known as the B-factor iterative test principle method where areas in a protein with high thermal fluctuations are identified from the X-ray crystal structures.…”

Combining computational and experimental screening for rapid optimization of protein properties

“…Multiple simultaneous mutations can be made, and novel sequences that are very different from wild type can be discovered. PDA has shown tremendous success in designing proteins with improved stability and conformational specificity (13,14,(24)(25)(26)(27)(28) and has even been used to engineer a catalytic site into a previously nonreactive protein (29).…”