|ENZYMOLOGY AND PROTEIN-LIGAND INTERACTIONS
Sequencing of the human genome, and those of numerous pathogens, has resulted in an explosion in the number of studies aimed at identifying potential drug targets. A key step in this effort, and one of the significant challenges, is to identify and characterize disease-associated proteins and enzymes, and, for the latter, to understand their specific mechanisms of action. This information is crucial for the discovery and optimization of lead molecules that target these proteins, as is the development of high through-put screening methods aimed at identifying substrates and inhibitors.
Enzyme kinetic parameters, such as the Michaelis-Menten constant Km and the maximum velocity Vmax, are commonly determined spectrophotometrically, requiring that the product of the enzymatic reaction exhibit strong absorption or fluorescence at a characteristic wavelength different from the reactants. However, many natural substrates do not provide the necessary chromophores. As a consequence, special chromophoric substrates must be used, or the product must be converted to a chromophore in another reaction via a coupling enzyme. Multi-step synthesis of the artificial substrate can be labor intensive and time consuming and the kinetic behavior of the synthetic chromophoric substrates may differ significantly from the native substrate. The presence of a coupling enzyme with additional substrates complicates the assay system and may interfere with the inhibition studies. Similarly, deconvoluting the different behaviors may be tedious, if not impossible.
We are developing direct, mass spectrometry-based assay methods that avoid these problems. We already see a clear advantage, since the approach is sensitive, requires no additional time or effort than an UV based method, and eliminates the need for a chromophore or radio-label. The major areas currently being investigated include:
- Enzyme immobilization and multiplex screening of combinatorial libraries of possible enzyme inhibitors.
- Determination of kinetic parameter such as Km, and kcat of specific enzymes as they relate to various substrates using a novel mass spectrometric method.
- Determination of Ki values for inhibitors identified in the screening process and simultaneous determination of mechanisms responsible for inhibition.
- Protein expression and unambiguous determination of the mechanisms (i.e. random, sequential, Ping-Pong) associated with various enzymes. Our new MS methods include isolating and identifying any intermediates using both digestion and stable isotope labeling techniques.
- Isolation and investigation of the enzyme-ligand non-covalent and covalent complexes and calculation of Kd values using gas-phase and solution phase data.
Our initial results on multiplex screening of enzyme inhibitors of pepsin and glutathione sulfotransferase have been followed by screening of combinatorial libraries of possible inhibitors of estrogen sulfotransferase, glcNAc-6-sulfotransferase (NODST) and hexokinase. One notable finding is that we have been able to identify potential inhibitors that otherwise would have been missed. The high resolution capabilities of the FTICR enable us to separate nominal mass isobars, thus allowing for the separation and identification of those compounds that bind to the enzyme while eliminating those that do not. This capability is critical because combinatorial libraries frequently contain compounds that have the same nominal mass but different exact mass. In several cases, we have discovered that one of the isobars inhibits the enzyme while the other does not. High resolution mass spectrometry is therefore paramount for this analysis.
We are also developing novel electrospray mass spectrometric (ESI-MS) assays that enable us to study substrate binding and the associated kinetic mechanisms for the enzymes NodST, glutathione sulfotransferase, phosphomannomutase and hexokinase. The mass spectrometric assay can be used to analyze the reaction using natural substrates without the need of a chromophore or radio-labels. A one-point normalization factor is generated to determine reaction velocities thus simplifying and substantially reducing the analysis time. The Km and kcat values of the enzymes are generated and similarly, Ki values for individual inhibitors are measured. The data clearly indicate whether the inhibitors are competitive or non-competitive. This technique can also be used to identify the specific kinetic mechanism of an enzyme and the pathways by which its complexes are formed. For example, in the case of NODST, the data indicate that the enzyme operates via a Ping-Pong mechanism and clearly rule out random/sequential mechanisms. We believe that we have identified the sulfated intermediate, which we have verified with enzymatic digestion and 34S labeling thus tracking the protein-SO4 intermediate by mass spectrometry.
Non-covalent complexes of the enzyme with both substrate and inhibitor are also being generated and analyzed using FTICR mass spectrometry and Kd values will be measured for both the substrate-enzyme and the inhibitor-enzyme complexes. The ratio of dissociation constants determined from the gas phase data matches that of the solution values thus suggesting that the solution complexes can be measured directly using mass spectrometry. This result may provide crucial additional verification that the enzyme folds properly and functions normally in the steps leading up to the mass spectrometric analysis. We anticipate that the significant progress weve already made in this area will lead to investigations of protein-protein interactions and thus will aid in answering important questions about this area of biochemistry. (This research is currently funded by NIH R01 GM63581)
The field of glycobiology is currently experiencing a rapid and dramatic surge of interest in the research community as evidenced by the recent review articles and books that cover basic carbohydrate chemistry. Clearly, there are many important biological functions performed by oligosaccharides. Several examples include protein glycosylation by glycolipid precursors, carbohydrate interaction in neural cell adhesion and bacterial meningitis, nitrogen fixation in legumes, and inhibition of antibody binding to various blood groups. These are but a few diverse examples of the impact that carbohydrates have on living systems. Structural elucidation of these biological molecules, in all their complexity, is therefore of utmost importance if we are to understand how these compounds affect cellular functions. It is my conviction that mass spectrometry will play the defining role in the area of structure determination of oligosaccharides as the field of glycomics progresses.
Research currently being conducted in my group includes the use of Fourier transform ion cyclotron resonance (FTICR) and ion trap mass spectrometry in combination with collision induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). These techniques are used in the development of novel methodology for the characterization of carbohydrate structure in a variety of biological systems. Specific goals include:
- Development of ion molecule reactions and metal-ligand complexation for the identification of specific sites of phosphorylation, sialylation, sulfation and stereoisomeric differentiation.
- Sequence analysis of heparan sulfate derived from the extracellular matrix of various healthy and diseased cells specifically as it relates to structure-function relationship.
- Investigation of mycobacteria cell wall constituents including development of methods for the analysis of mycolyl arbinogalactans (mAG) and lipoarabinomannans (LAM).
We have made significant progress in the structural elucidation of oligosaccharides using our metal-coordination technology, particularly as it applies to determination of stereochemistry using mass spectrometry. My group was the first to show that, through the use of metal coordination, carbohydrates retain their confirmation in the gas phase and because of this research I was awarded the Biemann Medal. However, there are still a number of basic carbohydrate building blocks for which there is no unambiguous MS method of analysis. In particular, the sulfated, phosphorylated and sialylated oligosaccharides continue to elude the structural biologist intent on knowing the specific sites of modification. Thus our current plans include the use of ion-molecule reactions as a tool for targeting the phosphorylated and sulfated oligomers. Preliminary data using pulsed gas reagents indicate that the ion-molecule approach enables specific functional groups to be reacted selectively and that the resultant products can be quantified. This methodology is being applied to relevant biomolecules found in a variety of strains of Haemophilus influenzae (HI) and Neisserria meningitidis (NM). These bacteria contain lipooligosaccharides (LOS) that are highly phosphorylated and which we know to be sialylated. Mass spectrometric analysis of these compounds is challenging due to the fact that phosphate and sialic acid linkages are labile resulting in loss of information as to composition and structure during ionization/activation.
We are also currently investigating the combined use of enzymatic digestion and novel mass spectrometry protocols for identification, quantification and sequencing of sulfated glycosaminoglycans (GAG), including the isomeric species. Although mass spectrometry has been used previously to analyze these complex oligomers, unambiguous identification and sequence characterization, including isomeric distinction, is still lacking. The goals of this project are to determine the identity of specific disaccharides and tetrasaccharides in heparin and HS, ascertain if their distribution is random, blocked, or sequential and ultimately to determine unambiguously which sites are sulfated and obtain specific sequence information. In combination with the MS technology, we are developing computer software to facilitate data interpretation and handling. Collaborations currently underway involve the analysis of pro-tumor and anti-tumor fragments of the extracellular maxtrix from healthy and diseased tissue as well as identification and investigations of the resulting GAGs that are differentially sulfated but for which no definite structure/composition is known.
Concurrent with the research described above, we have begun to address the analysis and characterization of the five membered ring saccharides, the arabinofuranoses. We have considerable experience in the analysis of hexose oligomers to serve as the basis for procedures specific to the pentose saccharides. Our attention was drawn to this class of carbohydrates because they are relevant to mycobacteria and its virulence. The challenges from the point of view of mass spectrometry lie in both the size and complexity of lipids that comprise the mycolyl arbinogalactan complex (mAG) and the lipoarabinomannan (LAM). In order to be able to investigate these structures fully, we have established collaborations with several investigators to gain access to various strains of Mycobacteria tuberculosis (MTB) and Mycobacteria smegmatis (MSG). Additionally, we have begun development of protocols to grow MSG in our laboratory. These strains will allow us to develop the appropriate metal-coordination methods with synthetic standards and then apply them to the mAG and LAM fractions.
The thrust of this research is to extend our methodology to the complete structural identification of complex oligosaccharides that are not amenable to current techniques. The ability to apply these methods to biologically isolated, relevant oligosaccharides is critical, as are rapid methods that involve minimal sample preparation, concentration, and time. We have also invested a considerable amount of time and resources to understanding the mechanisms of the developed gas phase reactions as well as defining the fundamental properties of the structure specific metal complexes. An understanding of the fundamental processes is critical to developing, extending and refining our methods. (This research is currently funded by NIH R01 GM47356)
ANALYSIS OF HUMAN RIBOSOME COMPLEXES BY MASS SPECTROMETRY
Mass Spectrometry is becoming very important in the field of structural biology and, as such, can provide answers to detailed questions about complex biomolecule structure and function. Combining key personnel and research resources of structural biologists and bioanalytical chemists presents the research community with new and innovative protocols for the analysis of non-covalent complexes. Integrated efforts between these two chemical biology groups can be critical to answering fundamental questions such as those specific to the role of ribosome-IRES binding and recognition. This is a joint project between the groups of Prof. Jennifer Doudna and Prof. Julie Leary.
The ribosome, the cellular catalyst of protein synthesis, is one of the largest and most complex biological macromolecules. We are currently involved in a collaborative research project to test the hypothesis that the ribosome and its two component subunits exist in multiple functional states in human cells. Preliminary evidence suggests that distinct forms of the ribosome, differing by one or a few component proteins, may play critical roles in the control of gene expression during viral infection. Upon infection by viruses such as hepatitis C, an internal ribosome entry site (IRES) RNA upstream of the viral messenger RNA binds to host cell ribosomes and recruits them for viral protein synthesis (1, 2). The molecular basis for IRES-ribosome binding and the possibility that only a subset of human ribosomes are competent to bind IRES elements will be explored in two ways. First, ribosomes will be purified from human cell lines and allowed to bind in vitro to internal ribosome entry site (IRES) RNA from hepatitis C virus (HCV). Ribosome-IRES complexes will be separated from ribosomes incapable of IRES recognition, and the components of each sample will be analyzed by electrospray ionization mass spectrometry (ESI-MS). To control for possible inactivation of ribosomes during purification, ribosome-IRES complexes formed during active IRES-mediated translation in cell extracts will be isolated and analyzed by ESI-MS, and the results compared to those obtained above.
Electrospray ionization has become the ionization technique of choice for analyzing non-covalent complexes by mass spectrometry. A 7T actively shielded Fourier Transform Ion Cyclotron Resonance mass spectrometer (FTICR-MS) will be used to generate and analyze the ribosome complexes. This instrument, which is capable of very high resolution and mass accuracy, has been modified specifically for non-covalent complexes. (We are in the process of applying for a Q-TOF type instrument, which is most widely used for non-covalent complexes and are currently discussing a gift of this instrument from Micromass-Waters.) A newly acquired nano-spray source for the FTICR-MS will also allow us to analyze very low concentrations of these complexes. Additionally, implementation of various dissociation methods (SORI-CID and IRMPD) will provide a mechanism for dissociating the complex and/or its component proteins to obtain additional information such as dissociation constants between the ribosome and IRES and/or between various protein-protein aggregates.
Pliminary experiments have begun which involve denaturing human 40 S ribosomes and analyzing the constituent proteins by LC-ESI-FTICR mass spectrometry. Data thus far collected indicate that of 20 proteins detected, all have been identified and in 8 cases we have detected various post-translational modifications. The ability to obtain high resolution accurate mass data has been critical for identifying these component proteins of the ribosome. Investigation and development of mass spectrometric methods specific to analysis of ribosome-IRES complexes will provide the scientific community with an important tool that can be used to answer many more complex questions in structural biology.
The results of this project will be significant in several ways. First, a molecular understanding of IRES RNA - ribosome interactions will illuminate the mechanism of IRES-mediated ribosome recruitment, a critical aspect of infection by many viruses. Furthermore, a comprehensive understanding of human ribosome composition and subtypes is key to a detailed understanding of the control of gene expression at the level of translation. Finally, technology developed in the course of this project will be valuable to the study of many macromolecular complexes that play central roles in the control of gene expression. We plan to extend the scope of this project to encompass a research effort targeting complexes that control translation in human cells, and will make available technology resources that will be of broad benefit to the bioanalytical community. (Funding for this project is pending).