Biochem Blogs

Biochemistry blog, science writing

The perfect marriage of crystallography and mass spectrometry: PI3K

Laura Edwards NCSU Biochemistry graduate student

Laura Edwards
I'm on ScienceSeeker-Microscope

 

Sorry for the cheesy title, but I’m getting married in a couple of weeks and it is all I can think about (oh, and science of course).  I have to admit that I chose a GREAT paper this time!: “Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors” which was published last year in PNAS (1).  It is a great example of how people can utilize HDX-MS when they want to understand how the structure of their protein changes as a result of it binding to something else.  A lot of times, crystallography cannot address this because the proteins are too big or too dynamic.  So when all else fails, turn to mass spec!

The Williams group in Cambridge has spent time pondering the question, “How is PI3Kγ’s activity shaped by association with its p101 adaptor subunit, lipid membranes and G-protein βγ heterodimers?”.  First off, let me tell you a little bit about PI3Kγ… this protein is a member of the Class 1B of phosphoinositide-3 kinases (PI3K), whose activation is driven by activation of G-protein coupled receptors (GPCRs) (whereas Class IA PI3K’s are activated by receptor tyrosine kinase (RTK) activation). It is known that upon PI3Kγ activation, the catalytic subunit, p110γ, associates with its regulatory subunit, p101, as well as to the G-protein βγ subunit, resulting in membrane translocation of PI3Kγ… however, the precise mechanisms of these events are unknown.  PI3Kγ has many roles in processes such as inflammation, cell migration, cardiac function and wound healing.  Increased levels of PI3K products have been associated with several forms of cancers, so this is an important protein to study (2).

PI3K pathways

PI3K pathways

Continue Reading >>

Highly unusual proteinaceous infectious agents probed by hydrogen/deuterium exchange

Bob Grinshpon

Graduate Student Bob Grinshpon
I'm on ScienceSeeker-Microscope

 

Prion proteins are implicated in a perplexing class of infectious diseases called transmissible spongiform encephalopathies (TSEs). Prion proteins are ubiquitous among mammals with roughly 90% sequence identity across species. TSEs include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle, AKA mad cow disease.  The disease ontology involves the conversion of the cellular prion protein (PrPc) to a misfolded conformation, (PrPsc), that  accumulates in amyloid-like aggregates and leads to neurodegeneration. The ‘protein only model’ is the currently accepted model that has recently been corroborated with data, and it suggests that the misfolded protein state itself is the only requirement for disease transmission.

The structure of the PrPc has been established (Figure 1), but there has been great difficulty in determining the structure of the rogue PrPsc conformer. Most approaches to probing the structure of ordered aggregates involve the introduction of specific probes, which is not applicable to samples derived from mammalian tissues.  However, hydrogen-deuterium exchange coupled with mass spectrometry analysis is an exception. H/D exchange takes advantage of the rapid exchange of backbone amide hydrogens within unstructured regions compared to the relatively slow exchange of systematically hydrogen bonded structures. In 2011, the Surewicz lab at Case Western Reserve published a Brief Communication in Nature Structural and Molecular Biology (1). The results of this paper offer some of the first spectroscopic insight into disease relevant conformational conversion of prion proteins.

Continue Reading >>

To Exchange or Not to Exchange?

Michael Goshe

Michael Goshe
I'm on ScienceSeeker-Microscope

 

To Exchange or Not to Exchange? That is the question — at least for the graduate students participating in our Proteins Journal Club this semester (my apologies to those members of the Shakespearean Journal Club — although I do like the musical version of Hamlet as performed on Gilligan’s Island).

 

 

 

 

As you may have surmised since this is a biochemistry blog, the topic is hydrogen exchange (HX) in which an amide proton (N-H) of a protein is exchanged for a deuteron (D) where kch is the chemical exchange rate constant and is a function of pH and temperature.

HD exchange reaction

HD exchange reaction

The propensity of an amide proton of an amino acid residue of a protein to deuterium exchange is related to its hydrogen bonding environment. If the amide proton is participating in a strong hydrogen bond with a carbonyl oxygen as occurs in an alpha-helix or beta-sheet, it will be more difficult to exchange with a deuteron than an amide proton that is part of a loop or a region of a protein that has a high degree of flexibility and exposure to solvent (Figure 1). The sites of exchange can be monitored by NMR or mass spectrometry analysis, thus allowing protein folding and structural dynamics to be measured by the accumulation of deuterium.

Continue Reading >>

The traveling biochemist: Mass Spectrometry in Vancouver

 

Kevin Blackburn

Kevin Blackburn

The 60th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics was held in Vancouver, British Colombia the week of May 19-May24 at the Vancouver Convention Center.  The meeting is the largest mass spectrometry meeting in the world and covers the full range of MS related topics, from instrument design to every imaginable application of mass spectrometry.

Vancouver

Vancouver

The city was fantastic, other than occasional light showers. As has been the case for the past 15 or so years, applications related to protein analysis, including protein structure and proteomics, comprised well over half of the presentations. The meeting was attended by approximately 6,000 scientists from across the globe, including researchers from industry and academia as well as a large contingent of vendors. Myself and two members of Michael Goshe’s lab, Laura Edwards and Fan Liu, attended the meeting.

Laura Edwards

Laura Edwards

This was Laura’s first ASMS meeting, and she was like a kid at Christmas!  She did, however, actually work on the first day of the meeting as an assistant, skillfully guiding meeting attendees to various meetings and short courses while wearing a neat red ASMS uniform and carrying an “Ask Me” sign!

 

 

Continue Reading >>

Discovering underneath a “MudPit”

 

Fan Liu

Fan Liu

What is referred to as “MudPit” here is not “a pit of mud” but a technique in the mass spectrometry field which ResearchBlogging.org stands for “multi-dimensional protein identification technology”, a very powerful approach that has been widely used since the year of its inception in 2001.

Many efficient technologies have been developed to reveal global behaviors of DNA (genome) and RNA (transcriptome), such as DNA-DNA interactions (through chromatin conformation capture on chip) and global gene expression (through transcriptome sequencing). However, the systematic study of proteins (proteome) lags behind the analytical DNA and RNA analysis.

Continue Reading >>

Another reason to love mass spectrometry

 

Laura Edwards NCSU Biochemistry graduate student

Laura Edwards

Many scientists out there want to know about the ResearchBlogging.org dynamics of a protein or how a protein binds to small molecules. But sometimes that information is hard to get using classical techniques such as X-ray crystallography or NMR.

Maybe your protein is too large (NMR won’t work) or maybe it just won’t crystallize. A mass spec technique called hydrogen/deuterium exchange mass spectrometry (HDX-MS) can be useful when you have almost given up and can’t think of anything else to try!

Continue Reading >>