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PUBLICATIONS: NEW


Endocannabinoid enzyme engineering: Soluble human thio-monoacylglycerol lipase (sol-S-hMGL).
Karageorgos I, Zvonok N, Janero DR, Vemuri VK, Shukla V, Wales TE, Engen JR, Makriyannis A.
ACS Chem Neurosci. 2012. In press. DOI: 10.1021/cn3000263

ABSTRACT
In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu169;Leu176)-to-Ser(Ser169;Ser176) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser122 with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser122 residue as compared to that of Cys122 in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys122 residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser122, and the mechanisms underlying hMGL inhibition by different classes of small molecules. 

Pubmed: too new


Hydrogen exchange mass spectrometry: Are we out of the quicksand?
Iacob RE, Engen JR.
 J Am Soc Mass Spectrom. 2012. Jun;23(6):1003-1010. Invited 'Critical Insight'.

ABSTRACT
Although the use of hydrogen exchange (HX) mass spectrometry (MS) to study proteins and protein conformation is now over 20 years old, the perception lingers that it still has “issues”.  Is this method, in fact, still in the quicksand with many remaining obstacles to overcome?  We do not think so.  This critical insight addresses the “issues” and explores several broad questions including: have the limitations of HX MS been surmounted and has HX MS achieved “indispensable” status in the pantheon of protein structural analysis tools. 
 
Pubmed: 22476891