Complex multiprotein

The results on the hydrolysis of partially methylated /3-casein by plasmin indicate that proteins radiomethylated to a low level can serve as substrates for trypsin-like enzymes and probably for proteinases in general. Because it is likely that methylation will interfere with enzymatic attack at lysine residues, the complete hydrolysis of /3-casein probably would not be possible. Studies on mastitic milk demonstrate the usefulness of 14C-methyl proteins for qualitative examination of protein hydrolysis in complex multiprotein systems where resolution and characterization of individual protein fragments is difficult. The requirements in such studies are the availability of pure samples of the proteins under investigation and a suitable technique for separating the radio-labeled protein from hydrolytic products. 

Most P450 systems reqture complex multiprotein electron transfer chains. The search for srutable redox proteins that can efficiently deliver the electrons to the heme or even con-... 

Peptides and proteins do not carry out their biological functions in isolation. Their active forms may be associated with metal ions, small molecules, macromolecules including proteins and DNA, and they may form complex multiprotein assemblies. Biological processes occur in aqueous solution in the presence of salts and other ions whereas MS is used to probe substances isolated in vacuo conditions that could be incompatible. Despite this, the use of MS to study these interactions is growing rapidly. MALDI, which ionizes directly from the solid phase, does not lend itself to studies that mirror solution conditions, but ESI, which abstracts ions directly from solution by rapid evaporation of nebulized droplets, can monitor noncovalent associations very successfully. 

It should be emphasized here that the four major complexes of the electron transport chain operate quite independently in the inner mitochondrial membrane. Each is a multiprotein aggregate maintained by numerous strong associations between peptides of the complex, but there is no evidence that the complexes associate with one another in the membrane. Measurements of the lateral diffusion rates of the four complexes, of coenzyme Q, and of cytochrome c in the inner mitochondrial membrane show that the rates differ considerably, indicating that these complexes do not move together in the membrane. Kinetic studies with reconstituted systems show that electron transport does not operate by means of connected sets of the four complexes. 

Apoptotic initiator caspases (caspase-2, -8, -9 and -10) constitute a subgroup of the caspase family. These caspases are the first to become proteolytically active in the apoptotic cascade. Their activation takes place in multiprotein complexes initiated by pro-apoptotic stimuli, such as TNFa, a-Fas, staurosporine. Once activated, they can process their substrates, which include the apoptotic executioner caspases. 

Multiprotein complex that catalyses ATP-dependent degradation of proteins tagged with ubiquitin. 

Very large Serine/Threonine kinases and the molecular Target of Rapamycin, a naturally occurring secondary metabolite, TOR proteins function within multiprotein complexes to couple cell growth and stress responses to environmental and developmental cues. 

Biochemical purification of TOR demonstrated that this protein functions as the catalytic component of two distinct multiprotein complexes known as TOR complex 1 (TORC1) and TOR complex 2 (TORC2). Like TOR, these complexes appear to have been structurally and functionally conserved form yeast to man. The mammalian equivalents are known as mTORCl and mTORC2. 

Finally, the binding of specific transcription factors to cognate DNA elements may result in disruption of nucleosomal structure. Many eukaryotic genes have multiple protein-binding DNA elements. The serial binding of transcription factors to these elements—in a combinatorial fashion—may either directly disrupt the structure of the nucleosome or prevent its re-formation or recruit, via protein-protein interactions, multiprotein coactivator complexes that have the ability to covalently modify or remodel nucleosomes. These reactions result in chromatin-level structural changes that in the end increase DNA accessibifity to other factors and the transcription machinery. 

Allen JR, SA Ensign (1997) Purification to homogeneity and reconstitution of the individual components of the epoxide carboxylase multiprotein enzyme complex from Xanthobacter strain Py2. J Biol Chem 272 32121-32128. 

Berger, I., Fitzgerald, D.J. and Richmond, T.J. (2004) Baculovirus expression system for heterologous multiprotein complexes. Nature Biotechnology, 22 (12), 1583-1587. 

In Vivo Deletion Analysis of the Architecture of a Multiprotein Complex of Translation Initiation Factors... 

It should be emphasized that the nature of all presented protocols is very general and, thus, their application for a comprehensive characterization of your favorite multiprotein complex (YFMPC) in yeast might require only minor modifications. The logical sequence of all required steps is schematically shown in Fig. 2.1. The initial large-scale Ni affinity isolation of eIF3 followed by mass spectrometry (MS) of its subunit composition has already been described (Asano et al, 2002), and methods for identification of protein-protein interactions such as yeast two-hybrid (Y2H) and in vitro glutathione-S-transferase (GST) pull-down analysis are presented in volume 429. This chapter focuses on a description of the small-scale one-step in vivo affinity purification techniques that were used to determine the effects of deletions and... 

Figure 2.1 Schematic illustrating the ideal sequence of all steps to be taken toward a comprehensive characterization of a multiprotein complex of interest.

Caufield, M.P., Horiuchi, S., Tai, P.C., and Davis, B.D. (1984) The 64-kilodalton membrane protein of Bacillus subtilis is also present as a multiprotein complex on membrane-free ribosomes. Biochemistry 81, 7772-7776. 

Fancy, D.A., Melcher, K., Johnston, S.A., and Kodadek, T. (1996) New chemistry for the study of multiprotein complexes The six-histidine tag as a receptor for a protein crosslinking reagent. Cbem. Biol. 3, 551-559. 

Jerng, H.H., Kunjilwar, K., and Pfaffinger, P.J. (2005) Multiprotein assembly of Kv4.2, KChIP3 and DPP10 produces ternary channel complexes with ISA-like properties./. Physiol. 568, 767-788. 

Ser/Thr-protein phosphatases are ubiquitous enzymes which constitute the catalytic domains of multiprotein complexes. They are responsible for the dephosphorylation of a range of phosphoproteins. Several protein phosphatases have been characterized by X-ray crystallography and display an active site structure similar to purple acid phosphatase. 

Protein kinases, in cooperation with other proteins, form multiprotein complexes which are susceptible to activation upon external agonist stimuli. According to different functions in cell-cycle regulation, the conformational changes are initiated by autophosphorylation and dimerization transmitted by the previously discussed second messengers cAMP, cGMP, IP3, PIP3, AA and DAG. 

S. G. N. Grant, M. C. Marshall, K.-L. Page, M. A. Cumiskey, and J. D. Armstrong. Synapse Proteomics of Multiprotein Complexes En Route from Genes to Nervous System Diseases. Hum. Mol. Gen., 14 (Review Issue 2)(2005) R225-R234. 

---