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The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.

The amyloid-& beta;(A & beta;) peptide is associated with the developmentof Alzheimer's disease and is known to form highly neurotoxicprefibrillar oligomeric aggregates, which are difficult to study dueto their transient, low-abundance, and heterogeneous nature. To obtainhigh-resolution information about oligomer structure and dynamicsas well as relative populations of assembly states, we here employa combination of native ion mobility mass spectrometry and moleculardynamics simulations. We find that the formation of A & beta; oligomersis dependent on the presence of a specific & beta;-hairpin motif inthe peptide sequence. Oligomers initially grow spherically but startto form extended linear aggregates at oligomeric states larger thanthose of the tetramer. The population of the extended oligomers couldbe notably increased by introducing an intramolecular disulfide bond,which prearranges the peptide in the hairpin conformation, therebypromoting oligomeric structures but preventing conversion into maturefibrils. Conversely, truncating one of the & beta;-strand-formingsegments of A & beta; decreased the hairpin propensity of the peptideand thus decreased the oligomer population, removed the formationof extended oligomers entirely, and decreased the aggregation propensityof the peptide. We thus propose that the observed extended oligomerstate is related to the formation of an antiparallel sheet state,which then nucleates into the amyloid state. These studies provideincreased mechanistic understanding of the earliest steps in A & beta;aggregation and suggest that inhibition of A & beta; folding into thehairpin conformation could be a viable strategy for reducing the amountof toxic oligomers.

Cell penetrating peptides (CPPs) are generally defined as short positively charged peptides, containing 5–30 amino acids. Based on their physicochemical properties, they are classified as three main groups, namely hydrophobic, amphipathic, and hydrophilic. They are capable of interacting with the cell membrane without inducing serious toxicity, and they can carry cargo molecules across the membrane. Cargo molecules could be different therapeutics which makes CPPs valuable in the field of drug delivery into living cells. Nowadays, CPPs are considered as potential parts of therapeutics against several diseases. Despite similarities in their primary structure, the interactions of CPPs with a cell membrane may vary a lot. This is even more complicated when the CPP is bound to the cargo molecule. The mechanism(s) of their cellular uptake and endosomal escape have not been completely resolved. Understanding the mechanism of membrane interaction will help us designing a CPP with enhanced, selective cargo delivery, hopefully resulting in better disease treatments. So far energy independent direct membrane penetration and energy-dependent endocytosis have been suggested as two main mechanisms of cellular entry for CPPs, and both may be applicable for the same CPP-complex, depending on the conditions. In order to understand which mechanism is associated with a particular CPP’s cellular uptake in a particular cell (sometimes including endosomal escape), different biological and biophysical methods and strategies have been applied. In this chapter, we will address several biophysical methods, such as fluorescence spectroscopy, circular dichroism (CD) spectroscopy, dynamic light scattering, and NMR. We also review different membrane model systems which are suitable for the biophysical studies. These include large unilamellar phospholipid vesicles (LUVs), which are the most commonly used in the lipid–peptide interaction studies. Detergent micelles and mixed micelles (bicelles) ar