; R o o t E n t r y F ~ SI @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l ~ SI ~ SI 4 @ C D E F G H I J K L F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; / @ * Index 1 ` , $ ` 9512711 Landry Chaperonins are a class of molecular chaperones characterized by a ring shaped oligomeric structure and the distinctive ability to use ATP to promote protein folding. Prokaryotic chaperonins usually are composed of two types of polypeptide, cpn60 and cpnl0, which in Escherichia coli are GroEL and GroES. GroEL has a weak ATPase activity, binds and stabilizes unfolded proteins, and releases them in the presence of ATP. GroES, also known as the co chaperonin, binds to GroEL in the presence of ATP, increases the cooperativity of the GroEL ATPase, and promotes ATP dependent discharge and folding of GroEL bound proteins. Since GroEL/GroES is necessary for folding in vivo of a large fraction of E. coli proteins, the mechanism of chaperonin action must not depend on specific features of substrate proteins. However, the bacteriophage T4 encoded co chaperonin, Gp31, is specifically required for GroEL dependent bacteriophage capsid assembly. The mechanism of chaperonin function is poorly understood, and how co chaperonins promote folding and whether they confer substrate specificity is presen tly unknown. This research addresses co chaperonin structure/function relationships and investigates how Gp31 specifically supports chaperonin function in bacteriophage T4 growth. Chaperonin assisted citrate synthase refolding will be evaluated for GroES in which the mobile loop has been specifically mutated to reduce or enhance GroEL binding. GroEL binding affinities will be measured by fluorescence techniques with GroES variants containing tryptophan. The effect on mobile loop dynamics and peptide conformation will be analyzed by nuclear magnetic resonance experiments. The affinity of GroES for GroEL will be compared to that of Gp31 for wild type and mutant GroEL. A genetic screen will be carried out to identify novel GroES mutants that have acquired the ability to replace Gp31 in T4 growth. These studies will illuminate mechanistic features of chaperonin function and identify determinants of chaperonin specificity for substrates and co chaperonins. This information will be useful for heterologous gene expression in biotechnological applications. %%% The folding of proteins into their active three dimensional structures is facilitated by a group of proteins called molecular chaperones. Several types of molecular chaperones are found in all organisms. One type of chaperone is composed of two ring shaped structures; and in the bacterium E. coli, they are called GroEL and GroES. GroEL and GroES bind to each other when they are assisting the folding of other proteins. GroEL alone can bind and release unfolded proteins, but GroES is essential for certain proteins to fold correctly. The importance and specificity of the GroES/GroEL interaction is shown by the chaperone requirements of the bacterial virus T4. This virus encodes its own version of GroES which is necessary for viral infection. This research will investigate how GroES directs the function of GroEL and why the viral version of GroES is necessary for viral infection. *** ....()()))()() Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT marcia steinberg marcia steinberg @ m 2QI @ S u m m a r y I n f o r m a t i o n ( B RI @ oSI @ `4< Microsoft Word 6.0 4 e = e D D D D D f D C p t t t t t t t #
