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Carbonic anhydrase (CA) is a thoroughly studied enzyme. Its primary role is the rapid interconversion of carbon dioxide and bicarbonate in the cells, where carbon dioxide is produced, and in the lungs, where it is released from the blood. At the same time, it regulates pH homeostasis. The inhibitory function of sulfonamides on CA was discovered some 80 years ago. There are numerous physiological-therapeutic conditions in which inhibitors of carbonic anhydrase have a positive effect, such as glaucoma, or act as diuretics. With the realization that several isoenzymes of carbonic anhydrase are associated with the development of several types of cancer, such as brain and breast cancer, the development of inhibitor drugs specific to those enzyme forms has exploded. We would like to highlight the breadth of research on the enzyme as well as draw the attention to some problems in recent published work on inhibitor discovery.

A mixture of explosives was analyzed by gas chromatography (GC) linked to ultraviolet (UV) spectrophotometry that enabled detection in the range of 178-330 nm. The gas-phase UV spectra of 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), ethylene glycol dinitrate (EGDN), glycerine trinitrate (NG, nitroglycerine), triacetone triperoxide (TATP), and pentaerythritol tetranitrate (PETN) were successfully recorded. The most interesting aspect of the current application is that it enabled simultaneous detection of both the target analyte and its decomposition products. At suitable elevated temperatures of the transfer line between the GC instrument and the UV detector, a partial decomposition was accomplished. Detection was made in real time and resulted in overlaid spectra of the mother compound and its decomposition product. Hence, the presented approach added another level to the qualitative identification of the explosives in comparison with traditional methods that relies only on the detection of the target analyte. As expected, the decomposition product of EGDN, NG, and PETN was NO, while TATP degraded to acetone. DNT and TNT did not exhibit any decomposition at the temperatures used.

Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of beta-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes., Funding Agencies|European Commission [FP7-ICT-2011-7, ERC StG 278242]; Goran Gustafsson Foundation; Swedish Alzheimer Foundation

More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 degrees C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 degrees C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer beta-strands I and VI of the beta-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817-8829) of amide hydrogen exchange at pH 7 and 25 degrees C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local beta-strands., Funding Agencies|Swedish Research Council [Dnr 2006-4253, Dnr 621-2012-5136]

The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.

The identification of novel enzymes for use in industrial biotechnology is an important goal in enzyme discovery. Most industrially relevant enzymes to date have been isolated from pure cultured microorganisms. For future discovery of novel enzymes this is however a major bottleneck since it is well established that only a small fraction of all microorganisms can be obtained in pure cultures. The possibility to identify enzymes directly from complete microbial communities would therefore give access to a huge number of novel enzyme candidates. Metaproteomics has hitherto mainly been used to understand ecosystem functions. We have instead used the dynamics of proteomics to develop a method based on “induced differential metaproteomics”, by which a desired enzyme activity is induced in a full microbial population and compared to a non-induced reference of the very same population. In a first example the goal was to induce, select and identify cellulases from a thermophilic methanogenic community. Out of several hundred detectable proteins in a 2D-DIGE experiment, 24 proteins could be identified as at least two-fold up-regulated upon induction. For some proteins spots, the cellulolytic activity was further validated by activity staining using 2D-zymography. Mass spectrometry analysis revealed that 21 out of the 24 up-regulated proteins are cellulases or associated to cellulolytic activity giving a remarkable hit-rate of 88%. This demonstrates the high efficiency and precision of the method, by which a much wider span of the microbial world can be scanned for novel and targeted enzymes.

The identification of novel enzymes for use in industrial biotechnology is an important goal in enzyme discovery. The need for novel biocatalysts for sustainable and efficient bioenergy production and the development of new biomaterials especially gives rise to new strategic opportunities of proteomic research. Most industrially relevant enzymes to date have been isolated from pure cultured microorganisms. It is however well established that only a small fraction of all existing microorganisms can be obtained in pure cultures, thus limiting the potential of finding novel enzymes. The possibility to identify valuable enzymes directly from complete microbial communities would therefore potentially give access to a huge number of novel enzyme candidates. Metaproteomics, or “the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time” has hitherto mainly been used to understand ecosystem function. In order to reach our goals we have instead used the dynamics of metaproteomics to develop a method based on “induced differential metaproteomics”, in which a desired enzyme activity is induced in a microbial population and compared to a non-induced reference of the very same population. In a first example the goal was to induce, select and identify cellulases from a methanogenic community, maintained in a biogas reactor at a metabolic steady-state in a chemically defined medium. Two aliquots were subtracted from the reactor, of which one was treated to induce cellulase activity. At the peak of cellulase activity and biogas production in the induced sample, proteins from the liquid phase of the two samples were prepared for 2D-DIGE of the extra-cellular proteins. Out of several hundred protein spots generated by the microbial community and visible in the 2D-DIGE experiment, 95 could be identified as up-regulated in the induced sample by image analysis, as compared to the references (thus representing potential cellulas

Two binder candidates 4-C37L34-B and 3-C15L8-B from a 16-membered set of 42-residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence-based screening assay. Two carbonic anhydrase isoforms with 60% homology exist in human blood with HCAI being present in five-to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16-membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4-C37L34-B bound HCAII with a K-D of 17 nm and HCAI with a K-D of 470 nm, that is, with a 30-fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two carbonic anhydrases were 60 and 390 nm, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.