Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchi.
Brier S, Maria G, Carginale V, Capasso A, Wu Y, Taylor RM, Borotto NB, Capasso C, Engen JR.
FEBS J. 2007, Dec;274(23) 6152-6166.

ABSTRACT
The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 °C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in E. coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than the mesophilic counterpart. The specificity of Antarctic fish pepsins was similar but not identical to pig pepsin, likely owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.
 
Pubmed: 17976195

Allosteric Loss-of-Function Mutations in HIV-1 Nef from a Long-term Non-progressor.
Trible RP, Emert-Sedlak L, Wales TE, Ayyavoo V, Engen JR, Smithgall TE.
J Mol Biol. 2007, Nov 16;374(1):121-129.
 
ABSTRACT
Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site allosterically influence Nef interactions with a key effector protein linked to AIDS progression.
 
Pubmed: 17920628

Probing Protein Interactions using Hydrogen-Deuterium Exchange Mass Spectrometry.
Weis DD, Kaveti S, Wu Y, Engen JR.
In “Mass Spectrometry of Protein Interactions”, 2007
ISBN: 978-0-471-79373-1, John Wiley & Sons. Kevin Downard, Editor.

Cover art: Inspired by Figure 3.3

CHAPTER 3 I. Introduction II. Hydrogen exchange background III. General HX MS method    a. Location information provided by HX MS    b. Revealing interactions by comparison IV. Interactions of proteins V. Examples    a. Conformational changes of proteins during binding    b. Protein:protein interactions    c. Protein:peptide interactions    d. Protein:small molecule interactions VI. Conclusions VII. References VIII. Acknowledgements IX. Figure legends X. Figures

ABSTRACT
Hydrogen-deuterium exchange in combination with mass spectrometry has become a powerful analytical tool for the investigation of protein interactions and protein dynamics. The technique is based on the exchange of protein amide hydrogens for deuterium when a protein or protein complex is placed in D2O solution. The isotopic exchange can be localized and quantified by measuring the mass increase with high resolution mass spectrometry. The fundamentals of H/D exchange MS are described along with its applications in various areas of protein chemistry. The method can provide valuable information about protein folding, about the conformational changes in proteins during binding or activation and inactivation, and can also be used to determine the dissociation constants of proteins in complexes. The application of hydrogen exchange is illustrated with recent experiments involving protein:protein interactions and protein interactions with small molecules.

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The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.
Chen S, Brier S, Smithgall TE, Engen JR.
Protein Sci. 2007 Apr;16(4):572-581.

ABSTRACT
The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the downregulated conformation.  Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity.  Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain.  Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution.  Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain.  Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions.  The observation that the Abl linker sequence exhibits SH3 binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases.  These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation that in Src kinases, where the downregulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.
 
Pubmed: 17327393

Functional characterization and conformational analysis of the Herpesvirus saimiri
Tip-C484 protein.
Mitchell JL, Trible RP, Emert-Sedlak LA, Weis DD, Lerner EC, Applen JJ, Sefton BM, Smithgall TE, Engen JR.
J Mol Biol. 2007 Mar 2;366(4):1282-1293.
 
ABSTRACT
Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific Src-family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of strongly activating Lck kinase activity and was multiply phosphorylated by Lck. Hydrogen exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogens became deuterated after only 10 seconds of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.
 
Pubmed: 17207813