Dagmar Ringe

The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein–protein, protein–small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein–protein interaction binding sites using heavy water (18O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software.

This chapter contains sections titled:
Introduction
Solvent Mapping
Characterization of Protein–Ligand Binding Sites
Functional Characterization of Proteins
Experimental Methods for Locating the Binding Sites of Organic Probe Molecules
Structures of Elastase in Nonaqueous Solvents
Organic Solvent Binding Sites
Other Solvent Mapping Experiments
Binding of Water Molecules to the Surface of a Protein
Internal Waters
Surface Waters
Conservation of Water Binding Sites
General Properties of Solvent and Water Molecules on the Protein
Computational Methods
Conclusion
Acknowledgments
References

Genomics, the study of the properties of genes and gene products on a whole-organism scale, is revolutionizing all aspects of biology. So powerful has knowledge of the complete nucleotide sequences of the genomes of whole organisms proven to be, that it has spawned a large family of progeny, each shifting the emphasis of their disciplines to discovery- driven (as opposed to hypothesis-driven) research: high-throughput, genome-scale data acquisition. Among the fields that have jumped onto the genomics bandwagon most rapidly is the field of structural biology. The painstaking determination of structures of individual proteins by laboratories that then spent years following-up that work by looking at structures of ligand complexes or mutants is being augmented by assembly-line production of structures for all of the proteins in a pathway or even a whole microbe, as rapidly as possible, with any follow-up work to be left to others. Structural genomics, as this effort is called, has as its stated goals the filling-in of the catalog of known protein folds and the assignment of function to gene products whose functions are not known (these may make up 40% of the gene products in a typical genome), by structural similarity to proteins of known function. How realistic are these expectations? What will be the impact on drug discovery and development? And what other tools are needed to realize the promise inherent in this richness of data?

While the E177S mutation in D-amino acid transaminase has a reduced ability to transaminate D-alanine, the efficiency of β-elimination of β-chloro-D-alanine remains almost intact. Interestingly, the latter reaction showed the appearance of an intermediate absorbing around 460 nm with this attenuated enzyme. The protein CD spectrum of the apo-enzyme of E177S resembled that of wild-type enzyme, but that of E 177K showed a negative elipticity around 280 nm.

This chapter contains sections titled:
Introduction
Apo‐DtxR crystal structure
Metal‐ion activation
Mechanism of metal‐ion activation
Nucleic acid recognition
DtxR homologs
Conclusions

This chapter discusses consumer's guide to protein crystallography. The atomic structures of large and small molecules can be determined by a technique called X-ray diffraction provided the molecule in question can be crystallized. Most of the three-dimensional structures of proteins, nucleic acids, and viruses that are known at present have been determined by X-ray crystallography. In favorable cases, the resolution of a crystallographic structure determination is such that the relative positions of all non-hydrogen atoms are known to a precision of a few tenths of an angstrom unit. There are two reasons for a non-specialist care about the details of this technique: first, the demand for structural information is so intense, and the competition to publish structures first has become so keen, that premature structure, incorrect in whole or in part, has appeared in the literature. Some of these have even found their way into the Protein Data Bank, the repository for protein three-dimensional structural information maintained at the Brookhaven National Laboratory. Second, structural information has become so central to enzymology, cell biology, and immunology that many people in those fields are likely to be involved in collaboration with a protein crystallographer at some point in their careers. The chapter focuses to teach this basic information.

The coordinate control of virulence determinants in pathogenic microorganisms is largely based upon the ability of these microbes to rapidly adapt to the environment presented by their host. Essentially all microbial pathogens have evolved specific mechanisms for the assimilation of sufficient concentrations of iron from their environment to support growth. It is remarkable that sensing the available concentration of iron provides not only a signal for siderophore expression, but also a regulatory signal for the expression of a wide variety of bacterial toxins and other virulence factors (reviewed in ref. 1). In Escherichia coli it is widely known that the coordinate regulation of iron-sensitive genes is mediated by Fur (ferric uptake regulator). Once it is activated by iron, Fur has been shown to function as a global regulatory element controlling the expression of regulons that are distributed throughout the E. coli chromosome. Moreover, it is now clear that the iron-mediated regulation of virulence genes in a number of Gram-negative pathogens is also coordinated by a family of closely related proteins that are homologous to Fur.

This chapter focuses on the structure-based design strategies employed to inhibit the HIV protease. The design strategy takes two general forms: (1) determination of crystal structures of complexes between the protease and compounds with known inhibitory potency to define characteristics leading to efficient binding and (2) theoretical approaches using the known crystal structure of the protease to predict structures of inhibitory compounds followed by experimental verification. It is difficult to find patterns that lead obviously to high affinity if the peptide-based HIV protease inhibitors are considered. The general principle that increasing the hydrophobicity of the inhibitor leads to tighter binding seems to be established, but the question of the role of symmetry remains unresolved. Putative transition state analogs are effective inhibitors because they add at least one strong hydrogen bond but studies of these compounds have not established the mechanism of the enzyme nor have they confirmed the hypothesis that their tight binding results from their resemblance to the activated complex.

Proteins are dynamic systems. Their dynamic properties can be divided into three broad classes: individual atomic fluctuations, collective motions of bonded and nonbonded neighbouring atoms, and ligand-induced conformational changes (Ringe and Petsko, 1985). The first two classes represent small-amplitude excursions around the equilibrium conformation of a protein; triggered conformational changes lead to the formation of a new average structure. Although the time-scale of individual and collective fluctuations is relatively short (10−13 to 10−9 sec), they can be studied by a variety of spectroscopic techniques and can be simulated computationally by molecular dynamics calculations (Karplus and McCammon, 1983). It has even proved possible to map the spatial distributions of these motions by X-ray crystallography, because they produce a spreading of the electron density around each atom, which may be modeled by various distribution functions (Petsko and Ringe, 1984; Ringe and Petsko, 1985). Triggered conformational changes have proven much more difficult to study in detail. Their time-scales are too long (10−6 to 101 sec) for simulation by simple molecular dynamics techniques. Moreover, since they produce a change in the equilibrium conformation of the protein, they involve crossing relatively large potential energy barriers. Theoretical methods for simulating barrier crossings are only just being developed, and they require detailed knowledge of both the initial and final states of the molecule, as well as of any intermediate structures that have a lifetime longer than that of a single atomic vibration (10−15 sec).

There has recently been a major revival of interest in serine protease structure, mechanism and inhibition. Recognition that this class of enzymes plays an essential role in such diverse and important processes as cellular regulation, viral infection, blood coagulation, and diseases like emphysema has led to new efforts to control their reactivites. Because serine proteases are ubquitous, but highly specific, they pose special problems as targets for inhibitor design. A “generic” protease inhibitor that bases its reactivity solely on the nucleophilicity of the active site serine hydroxyl will inactivate too many essential proteases as well as the target enzyme; phenylmethanesulfonyl fluoride is such a compound, and is highly toxic. Consequently, the most fruitful approach would be to use the natural specificity of the protease to direct the inhibitor to only the desired target. This specificity resides in a set of specificity sub-sites, some distance from the reactive serine and extending away from it in two opposite directions. Naturally occurring protein protease inhibitors derive their specificity from a number of interactions between inhibitor and enzyme, which are not restricted to the catalytic site or primary specificity site.