Search

One of the goals of proteomics is to predict the functions of all proteins. Many protein functions involve interactions with other biological molecules. Some of the most important interactions are those of proteins with small molecule ligands. Several experimental and computational approaches have been proposed previously to predict the locations of ligand binding regions of proteins. The aim of this thesis is to test a new approach to identify the ligand binding regions on the surface of X-ray crystal structures of proteins with unknown functions. The hypothesis that was tested was that X-ray crystallographic B-factors, also known as temperature factors, of atoms located on the surface of a protein X-ray crystal structure, in combination with information about the relative sizes of surface pockets, can help in identifying the functional regions of proteins. We used as our test set of protein structures a non-redundant dataset of 328 proteins for which X-ray crystal structures have been solved for both ligand-bound and unbound forms. The main steps involved identifying the surface pocket that, out of the ten largest surface pockets, contains the largest number of atoms with low B-factor values, and then checking to see if that pocket corresponds to the known ligand-binding pocket. If so, the crystallographic B-factor values could be used as a parameter to aid in predicting the ligand-binding regions of many uncharacterized proteins. For the majority of proteins in our study, that is, for 66% of the proteins in our working dataset, the proposed methodology was able to identify correctly the ligand-binding pockets in the protein crystal structures of the unliganded forms of the proteins.

Cell surface proteins of bacterial pathogens play key roles in invasion and virulence. Colonization of the host requires adhesion of the bacterium to host tissues, so some surface proteins bind to proteins in the extracellular matrix or directly to host cells. Others bind to plasminogen, which, when converted to the active protease plasmin, aids in degradation and invasion of host tissues. Surprisingly, a growing number of the cell surface proteins that bind to host cells, extracellular matrix, or plasminogen were previously identified as intracellular enzymes or chaperones, and we refer to them as intracellular/surface moonlighting proteins. Moonlighting proteins are a subset of multifunctional proteins in which multiple biochemical or biophysical functions in one polypeptide chain are not due to gene fusions, families of homologous proteins, promiscuous enzyme activity or pleiotropic effects.It is not known how most intracellular/cell surface moonlighting proteins are secreted. These proteins do not possess signal peptides for secretion by the canonical Sec pathway. Their secretion may involve a novel version of another known secretion pathway or it may involve an as yet unknown secretion pathway. In addition, they do not contain sequence motifs known to be involved in attachment to the cell surface. This could also involve a new version of a known mechanism or it may involve an as yet unknown mechanism. With the increasing problem of antibiotic resistance, new targets for inhibiting bacterial infection and virulence are needed. Understanding how intracellular/cell surface moonlighting proteins are targeted to the surface of a pathogen might lead to a method to decrease the ability of bacteria to bind to and degrade host tissues and could provide new targets for developing therapeutics to treat infections.

Moonlighting proteins comprise an interesting subset of multifunctional proteins in which the two functions are found in a single polypeptide chain. They do not include proteins that are multifunctional due to gene fusions, families of homologous proteins, splice variants, or promiscuous enzyme activities. Moonlighting proteins include several different kinds of enzymes and other proteins and different combinations of functions. Recent crystal structures of some moonlighting proteins have provided clues to the molecular mechanisms of one or both functions, and in some cases how a protein can switch between functions. We are preparing a curated, searchable database that contains information about the sequences, structures, and functions for known moonlighting proteins.

Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms.