The Crystal Structure of Affinity-matured Human Growth Hormone at 2 Å Resolution

Ultsch, Mark; Somers, W.S.; Kossiakoff, A.A.; Vos, A.

doi:10.1006/jmbi.1994.1135

Cited by 107 publications

(61 citation statements)

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“…1). The second derivative spectrum of the native protein had a dominant band at 1654 cm Ϫ1 for ␣-helix, consistent with the 4-␣-helix bundle structure of rhGH (29). The spectrum for the protein aggregated and precipitated in buffer had a decrease in intensity of the ␣-helix band, which was compensated for by bands at 1627 and 1695 cm Ϫ1 .…”

Section: Effects Of Aggregation Conditions On Structure Of Aggregatessupporting

confidence: 58%

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

John¹,

Carpenter²,

Balny³

et al. 2001

Journal of Biological Chemistry

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Two different types of insoluble, non-native aggregates of recombinant human growth hormone were formed by agitation in buffer or buffer containing 0.75 M guanidine HCl (GdnHCl) and characterized by infrared and second derivative UV spectroscopies. The degree of secondary structural perturbation was greater in the aggregates formed in 0.75 M GdnHCl. Both aggregate types were dissolved and refolded using high hydrostatic pressures in combination with either elevated temperature or non-denaturing levels of guanidine HCl or urea. The effects of a range of temperature, pressure, and chaotrope concentrations were tested and led to optimal conditions that approached 100% yield of native protein. The aggregates formed in 0.75 M GdnHCl required higher concentrations of urea or GdnHCl, or higher temperature or pressure for a yield equivalent to that for aggregates formed in buffer alone. Investigation of the effects of pressure, temperature, and chaotrope on unfolding of rhGH documented that under conditions used for optimal high pressure disaggregation and refolding, the native state is greatly favored thermodynamically (e.g. 25 kJ/mol at 2000 bar and 0.75 M GdnHCl). Dissolution of aggregates under pressure is a kinetically limited process. Comparison of refolding yields in GdnHCl and urea solutions suggest that pressure effects on electrostatic interactions do not dominate pressure effects on disaggregation. We suggest that non-native hydrogen bonds between protein molecules within aggregates of recombinant human growth hormone are responsible for the rate-limiting kinetic barrier in pressure-induced disaggregation.

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Section: Effects Of Aggregation Conditions On Structure Of Aggregatessupporting

confidence: 58%

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

John¹,

Carpenter²,

Balny³

et al. 2001

Journal of Biological Chemistry

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“…Furthermore, formation of the more C-terminal of these helices allows the double titration of His 7~ to be explained. These helices are also present in the soluble form of an hGH mutant [17]. In hGH they are involved in receptor binding [14] and, if this is indeed a conserved feature, they may have similar functions in G-CSF and LIF.…”

Section: Discussionmentioning

confidence: 99%

“…Because this helix is not seen in porcine GH [16] it has been suggested that in hGH it may form as a consequence of receptor binding [14] and that the similarity with hG-CSF may not be real [6]. Recently, however, it has been observed in a crystal structure of an hGH mutant that is not receptor bound [17]. The remainder of the AB loop was taken from a search of structures in the Protein Data Bank [18].…”

Section: Model Buildingmentioning

confidence: 99%

Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor

et al. 1994

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Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with ~H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pKa values of which range from 3.6 to 7.4. These pKa values were assigned to individual histidine residues from NMR studies on a series of His --* Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.

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“…Concurrently, the C-terminal end of helix-4 moves 1.1 Å closer to helix-1. The space introduced by the absence of the Phe side chain is filled by a water molecule that is totally buried in the hydrophobic core H-bonding to two carbonyl groups, A10 and L177 (32).…”

Section: H/d-ex Analysis Of Hghvmentioning

confidence: 99%

The Role of Protein Dynamics in Increasing Binding Affinity for an Engineered Protein−Protein Interaction Established by H/D Exchange Mass Spectrometry

et al. 2006

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It is generally accepted that protein and solvation dynamics play fundamental roles in the mechanisms of protein-protein binding; however, assessing their contribution meaningfully has not been straightforward. Here, hydrogen/deuterium exchange mass spectrometry (H/D-Ex) was employed to assess the role of dynamics for a high-affinity human growth hormone variant (hGHv) and the wild-type growth hormone (wt-hGH) each binding to the extracellular domain of their receptor (hGHbp). Comparative analysis of the transient fluctuations in the bound and unbound states revealed that helix-1 of hGHv undergoes significant transient unfolding in its unbound state, a characteristic that was not found in wthGH or apparent in the temperature factor data from the X-ray analysis of the unbound hGHv structure. In addition, upon hormone binding, an overall increase in stability was observed for the -sheet structure of hGHbp which included sites distant from the binding interface. On the basis of the stability, binding kinetics, and thermodynamic data presented, the increase in the binding free energy of hGHv is primarily generated by factors that appear to increase the energy of the unbound state relative to the free energy of the bound complex. This implies that an alternate route to engineer new interactions aiming to increase protein-protein association energies may be achieved by introducing certain mutations that destabilize one of the interacting molecules without destabilizing the resulting bound complex. Importantly, although the hGHv molecule is less stable than its wt-hGH counterpart, its resulting active ternary complex with two copies of hGHbp has comparable stability to the wt complex.A principal goal in structural biophysics is to develop general rules for structure-function relationships that govern protein-protein interactions, ultimately to quantify the contribution of individual local interactions to the overall binding energy. In this regard, there is a growing appreciation that essentially all interactions are context-dependent, and factors such as solvation and dynamics play critical, if not dominant, roles in forming most surface-to-surface contacts. Because solvation and dynamics are inherently difficult phenomena to quantify experimentally, their influence many times is invoked in a very general way, especially when structure-based rationales fail to explain biophysical data.The interaction between a high-affinity variant of human growth hormone (hGH) and the extracellular domain of its cognate receptor (hGHbp) 1 represents one of the cases where the binding properties are difficult to rationalize based on general structure-function principals. This variant (hGHv) was produced by phage display mutagenesis and binds through Site-1 to hGHbp with a K d < 10 pM, which constitutes a ∼400-fold (∼3 kcal/mol) increased affinity over the wild-type hormone (wt-hGH), K d ∼1 nM (1-3). It contains 15 mutations in its Site-1 binding interface and retains its full biological activity (4).An unanticipated finding w...

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The Crystal Structure of Affinity-matured Human Growth Hormone at 2 Å Resolution

Cited by 107 publications

References 0 publications

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor

The Role of Protein Dynamics in Increasing Binding Affinity for an Engineered Protein−Protein Interaction Established by H/D Exchange Mass Spectrometry

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The Crystal Structure of Affinity-matured Human Growth Hormone at 2 Å Resolution

Cited by 107 publications

References 0 publications

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

High Pressure Refolding of Recombinant Human Growth Hormone from Insoluble Aggregates

Homology modelling and 1H NMR studies of human leukaemia inhibitory factor

The Role of Protein Dynamics in Increasing Binding Affinity for an Engineered Protein−Protein Interaction Established by H/D Exchange Mass Spectrometry

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Homology modelling and ¹H NMR studies of human leukaemia inhibitory factor