The Hendrix lab is investigating how proteins work, and in particular how proteins interact with each other to assemble into an ordered biological structure. We use bacteriophages as experimental subjects because these viruses provide a system that is almost unequaled in the ease with which we can apply a wide array of experimental approaches (molecular genetics, biochemistry, biophysics, electron microscopy, structural biology) to tackle sophisticated questions about how proteins interact during assembly.
An important advance in our understanding of virus assembly that has come out of recent studies is that viral proteins go through a complex series of transitions (covalent and conformational changes) as assembly proceeds. In this view of assembly, finding their correct place in the growing structure is only the first step for the protein subunits: once they are in place they must flex, wiggle, and adjust their contacts with neighbors to progressively strengthen or otherwise modulate the properties of the structure as a whole. As we learn more, virus assembly comes increasingly to resemble an elaborately choreographed organic ballet. Our lab is studying virus assembly by studying individual examples of transitions in protein structure that take place during assembly of bacteriophages; we are also working to understand how these individual steps fit together in the overall logic of the assembly ballet. The principles we are learning describing how proteins interact to build a biological structure are applicable to many other biological systems in addition to viruses--from protein complexes that regulate gene expression to cytoskeletons--including those for which direct experimentation to address these questions is prohibitively difficult.
Head assembly of bacteriophage HK97.
HK97 is a close relative of the well known bacteriophage lambda with a particularly informative head assembly pathway. We are studying the structures of the various capsid precursors on this pathway by cryo-electron microscopy and X-ray crystallography. We can carry out most of the steps in the pathway in vitro, allowing us to study the detailed biochemical and biophysical properties of each reaction (including an unusual autocatalytic covalent crosslinking of all the head subunits). We have determined the DNA sequence of the 40 KB phage genome, which makes it easy to design and construct mutants that allow detailed dissection of each step of the pathway.
A portion of the high resolution structure of the bacteriophage HK97 capsid, determined by x-ray crystallography (see the paper by Wikoff et al.). The picture shows an area of the structure around one of the 3-fold symmetry axes; it includes portions of 9 different copies of the 420 identical protein subunits that make up the structure. In addition to backbone traces of the subunits, it shows 3 of the 420 inter-subunit covalent bonds (yellow amino acid side chains) that link all the subunits of the capsid into the fabulous chainmail topology. (This picture is a stereo pair; to see it in 3 dimensions, stare through the picture as if looking into the distance until the images from each eye merge in the middle.) The structure determination was carried out by our amazing collaborators Bill Wikoff and Jack Johnson at The Scripps Research Institute.
Assembly of the bacteriophage lambda tail.
The tail of phage lambda lambda is composed of a tail tip containing the fiber that interacts with the host bacterium and a long shaft composed mostly of a single tail protein. We have recently shown that a pair of chaperone proteins are required for the assembly of the long tail shaft and are currently exploring the details of the mechanism of the assembly of the tail to the correct length.
The Bacteriophage Genome Project.
In collaboration with Graham Hatfull and other members of the Pittsburgh Bacteriophage Institute, we have begun a project to determine the genomic sequences of a few dozen bacteriophages. We are comparing the sequences we produce to each other and to sequences in the databases in order to learn about mechanisms of virus evolution and the genetic structures of phage populations. The sequences of head and tail fiber genes of new phages are also of direct relevance to our studies of these aspects of HK97 and lambda biology. We are using DNA sequencing and DNA array technologies to compare the genes in this collection of phages with those of domesticated phages and those of other natural phage populations. The Phage Genome Project is a collaboration between our lab and those of Graham Hatfull and Jeffrey Lawrence.