Dr. Mohiuddin Kabir

Curriculum Vitae:

Two of the fundamental questions expressed in the Astrobiology Roadmap are “How does life begin and evolve?” and “Does life exist elsewhere in the universe?” Understanding the distribution of complex organic molecules associated with living things is essential to answering both of these questions. Microbiologist Mohiuddin Kabir is working with his NASA partner to develop a new technology for measuring the abundance of an array of organic molecules in complex mixtures with stereochemical specificity without chemical modification. Various instruments are under development to search for organic amines, amino acids and polycyclic aromatic hydrocarbons in extraterrestrial environments. The most mature of these is based on gas chromatography mass spectrometry (GCMS). This technology is at the heart of an instrument suite called SAM (Sample Analysis at Mars) that is part of the Mars Science Laboratory mission.

In GCMS instrumentation samples must be volatilized, either by pyrolysis or by chemical deriviatization. Pyrolysis breaks down complex molecules into smaller units that are analyzable but stereochemistry cannot be resolved. Derivatization reacts analytes with chemicals that increase their volatility, enabling GCMS analysis. Under appropriate conditions, stereochemistry of derivatized molecules can be resolved by GCMS. GCMS separations are, however, sometimes subject to degeneracy when multiple compounds have the same mobility and mass. This is particularly troublesome when the sample is not well characterized as would be the case in extraterrestrial environments. Uncharacterized chemical reactivity in the sample may also interfere with derivatization chemistry. The UREY instrument, planned for inclusion in the ESA ExoMars mission, contains the Mars Organic Analyzer (MOA) for the purpose of analyzing amines and amino acids on Mars. This instrument is based on micro capillary electrophoresis (µCE) combined with preparation of the sample by water extraction and chemical derivatization. The derivatization in this case adds a fluorescent dye to the analyte to facilitate detection. This instrument is extremely sensitive but has limitations similar to GCMS in that there is some risk that unknown compounds in the sample will either obscure the capillary electrophoresis results or interfere with the derivatization chemistry. Furthermore, the current derivatization chemistry only reacts with amines, leaving the instrument unable to detect sugars and other polyols that lack an amine. To overcome the problems associated with existing technologies, Mohiuddin Kabir and his NASA partner are developing protein based biosensors in order to detect organic amines, amino acids and polycyclic aromatic hydrocarbons in extraterrestrial environments that does not require the samples to be modified.

Projects

Protein Engineering of Bacterial Periplasmic Proteins

NNX07AC09A

It is generally agreed that organic molecules such as amino acids and carbohydrates are likely markers for extraterrestrial life as well as important components of a prebiotic environment. An important feature of amino acids and carbohydrates is that they both have chiral centers. While abiotic processes generally do not exhibit stereochemical selectivity, biological processes are highly selective. For example, all known terrestrial protein biosynthesis is based on L-amino acids, while the inverse stereochemistry D-amino acids occupy only specific niches in nature. Carbohydrates, such as the five and six carbon sugars and sugar alcohols that are ubiquitous in terrestrial biology, have numerous chiral centers. Amino acids and sugar alcohols are both known to exist in extraterrestrial environments and stereochemistry is the key aspect for distinguishing between abiotic and biotic origin. Amino acids of abiotic origin, such as those observed in extracts from the Murchison meteorite, are generally nearly racemic mixtures with small enantiomeric excesses in a few cases, while it is widely thought that living organisms are necessarily stereoselective. Among carbohydrates detected in Murchison meteorite extracts, there is little enantiomer selectivity, while living organisms favor a small number of specific enantiomers. Therefore, discovery of extraterrestrial organic compounds with large enantiomeric excesses would be a strong indication of extant life. On the other hand, near racemic mixtures could represent a pre-biotic environment. Since pure enantiomers racemize over time, intermediate degrees of enantiomeric enrichment would argue for dormant or extinct life.

For robust and definitive identification of selected biomarkers, we are developing technology to specifically detect an array of biologically relevant organic molecules, including their stereochemistry, without separation or chemical derivatization. This sensor technology is based on thermophilic bacterial periplasmic binding proteins (PBPs) that perform a sensing function in living cells. PBPs comprise a family of proteins in gram-negative bacteria involved in uptake of nutrients and other small molecules from the extracellular space. A variety of PBP ligands are known, including amino acids, peptides, simple and complex sugars, inorganic ions and metals, and each member of the family binds its ligand with high affinity and specificity. Two globular lobes surrounding a ligand binding site, and connected by a flexible hinge, form a conserved structural motif which is common to most PBPs. The family also exhibits a conserved “venus fly-trap” conformational change upon ligand binding, in which a hinge-twist motion brings the two lobes closer together. The ability to target a variety of diverse ligands combined with the specificity and dramatic structural change associated with ligand binding have attracted us in building sensing devices for industry, biology, medicine and astrobiology.

In order to create such biosensors with a large and consistent change in optical properties based on engineering of PBPs, our approach employs fluorescence resonance energy transfer (FRET). FRET is a nonradiative energy transfer from an excited donor to an acceptor fluorophore. For this transfer to take place, the excitation spectrum of the donor must overlap with the absorbance of the acceptor. The efficiency of the transfer is directly dependant on the distance between donor and acceptor. For each donor-acceptor combination the distance dependence is characterized by Förster distance (rf) at which transfer is 50% efficient. Moving donor and acceptor closer than rf results in increasing efficiency until a maximum is reached, while moving donor and acceptor further reduces efficiency until transfer is zero. We have exploited these phenomenon with PBPs by placing donor and acceptor fluorophores on the two domains of the protein as illustrated below.

Upon ligand binding, the two domains are brought closer along with the attached fluorophores, resulting in increased energy transfer. The signal from such a sensor can easily be measured using the ratio of donor emission intensity to acceptor emission intensity. This strategy for inducing a fluorescence signal change is general for all of the binding proteins and requires less detailed structural information than the use of environmentally sensitive fluorophores. It is also highly desirable that the trial and error approach can be avoided that is necessary when placing environmentally sensitive fluorophores. Another important advantage of FRET is that by measuring a ratio between emission intensity at two wavelengths, variations in light intensity and protein concentration do not contribute any error to the final signal.