Tracking a protein following dissociation from a protein–protein complex using a split SNAP-tag system

Protein–protein interactions (PPIs) are important for various biological processes in living cells. Several methods have been developed for the visualization of PPIs in vivo; however, these methods are unsuitable for visualization of post-PPI events such as dissociation and translocation. In this study, we applied a split SNAP-tag system for the visualization of post-PPI events. This method enabled tracking of the protein following dissociation from the protein–protein complex. Thus, the split SNAP-tag system should prove to be a useful tool for visualization of post-PPI events.     

Protein tyrosine phosphatase interacting protein 51—a jack-of-all-trades protein

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) interacts both in vitro and in vivo with PTP1B, a protein tyrosine phosphatase involved in cellular regulation. PTPIP51 is known to be expressed in many different types of tissues. It is involved in cellular processes such as proliferation, differentiation and apoptosis. Nevertheless, the exact cellular function of PTPIP51 is still unknown. The present review summarizes our current knowledge of the PTPIP51 gene and its mRNA and protein structure.     

Is NSF a fusion protein?

N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase required for vesicular transport throughout the constitutive secretory and endocytic pathways. Recently, NSF has also been implicated in regulated exocytosis in synapses--based on SNAP-mediated binding in vitro to a complex of neurotoxin substrates (termed 'SNAREs'). This work has generated an hypothesis in which the interaction of SNAREs (SNAP receptors) on the vesicle membrane with those on the target membrane forms a docking complex to which SNAPs bind, thus allowing NSF to bind and elicit membrane fusion. However, current evidence supports an earlier, pre-fusion role for NSF. We speculate that this role may be as a molecular chaperone for the membrane docking/fusion machinery.     

Fibronectin: a chromatin-associated protein?

We have previously reported that chromatin preparations from human cultured fibroblasts contain a single homologous serum protein. In this paper we present evidence, based on immunological identity and physicochemical properties, that this serum protein is fibronectin. Furthermore, using a radioimmunoassay system, we have estimated that fibronectin represents about 0.7% of the total protein in both chromatin preparations and whole fibroblasts. Using a nitrocellulose filter assay system, we also show that fibronectin is a DNA-binding protein having an equilibrium constant of 4.6 x 10(-6) M. Equilibrium competition experiments have demonstrated that fibronectin has the ability to differentiate among nucleotides, indicating that fibronectin-DNA interaction is at least partially specific, and that a minimum polymer length of 12-18 nucleotides is required for effective binding to occur. Fibronectin has been isolated readily from plasma using DNA-affinity chromatography. We do not have direct evidence that fibronectin is an actual nonhistone chromosomal protein, but fibronectin is a DNA-binding protein (at least under in vitro assay conditions) and appears to be a normal constituent of chromatin as chromatin is currently isolated from cell nuclei.     

Over-producing soluble protein complex and validating protein–protein interaction through a new bacterial co-expression system

Many proteins exert their functions through a protein complex and protein–protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein–protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein–protein interaction of interest in a wide range of biological organisms.     

Protein microarrays as a discovery tool for studying protein–protein interactions

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein–protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein–protein interaction studies.     

ModuleSearch: finding functional modules in a protein–protein interaction network

Many biological processes are performed by a group of proteins rather than by individual proteins. Proteins involved in the same biological process often form a densely connected sub-graph in a protein–protein interaction network. Therefore, finding a dense sub-graph provides useful information to predict the function or protein complex of uncharacterised proteins in the sub-graph. We developed a heuristic algorithm that finds functional modules in a protein–protein interaction network and visualises the modules. The algorithm has been implemented in a platform-independent, standalone program called ModuleSearch. In an interaction network of yeast proteins, ModuleSearch found 366 overlapping modules. Of the modules, 71% have a function shared by more than half the proteins in the module and 58% have a function shared by all proteins in the module. Comparison of ModuleSearch with other programs shows that ModuleSearch finds more sub-graphs than most other programs, yet a higher proportion of the sub-graphs correspond to known functional modules. ModuleSearch and sample data are freely available to academics at http://bclab.inha.ac.kr/ModuleSearch.     

Μyospryn: a multifunctional desmin-associated protein

Desmin, the muscle-specific intermediate filament protein, forms a 3D scaffold that links the contractile apparatus to the costameres of plasma membrane, intercalated disks, the nucleus, and also other membranous organelles. The cellular scaffold formed by desmin and its binding partners might be implicated in signaling and trafficking processes, vital mechanisms for the survival of the mammalian cell. One novel desmin-associated protein is the tripartite motif-like protein myospryn. Myospryn was initially identified as an associated partner to the biogenesis of lysosome-related organelles complex 1 protein dysbindin, implicating its potential involvement in vesicle trafficking and organelle biogenesis and/or positioning. Myospryn is also an A kinase anchoring protein, raising the possibility that together with desmin and other cytoskeletal and signaling proteins, it could participate in the subcellular targeting of protein kinase A activity in striated muscle. As with desmin, different members of this scaffold might play a crucial role in the pathogenesis of muscle disease, since any disturbance in these highly coordinated signaling pathways is expected to compromise efficient maintenance of structure–function integrity of muscle and lead to different cardiac and skeletal myopathies.     

Topology and weights in a protein domain interaction network – a novel way to predict protein interactions

Background

While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well.

Results

We consider a web of interactions between protein domains of the Protein Family database (PFAM), which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly.Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation.

Conclusion

Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we show a simple way to predict potential protein interactions by utilizing expectation scores of single domain interactions.

     

Protein dynamics of a β-sheet protein

Rhodnius prolixus Nitrophorin 4 (abbreviated NP4) is an almost pure β-sheet heme protein. Its dynamics is investigated by X-ray structure determination at eight different temperatures from 122 to 304 K and by means of Mössbauer spectroscopy. A comparison of this β-sheet protein with the pure α-helical protein myoglobin (abbreviated Mbmet) is performed. The mean square displacement derived from the Mössbauer spectra increases linearly with temperature below a characteristic temperature T _c. It is about 10 K larger than that of myoglobin. Above T _c the mean square displacements increase dramatically. The Mössbauer spectra are analyzed by a two state model. The increased mean square displacements are caused by very slow motions occurring on a time scale faster than 140 ns. With respect to these motions NP4 shows the same protein specific modes as Mbmet. There is, however, a difference in the fast vibration regime. The B values found in the X-ray structures vary linearly over the entire temperature range. The mean square displacements in NP4 increase with slopes which are 60% larger than those observed for Mbmet. This indicates that nitrophorin has a larger structural distribution which makes it more flexible than myoglobin.     

Simulation of protein diffusion: a sensitive probe of protein–solvent interactions

Aqueous solutions of Candida antarctica lipase B (CALB) were simulated considering three different water models (SPC/E, TIP3P, TIP4P) by a series of molecular dynamics (MD) simulations of three different box sizes (L = 9, 14, and 19 nm) to determine the diffusion coefficient, the water viscosity and the protein density. The protein–water systems were equilibrated for 500 ns, followed by 100 ns production runs which were analysed. The diffusional properties of CALB were characterized by the Stokes radius (R_S), which was derived from the diffusion coefficient and the viscosity. R_S was compared to the geometric radius (R_G) of CALB, which was derived from the protein density. R_S and R_G differed by 0.27 nm for SPC/E and by 0.40 and 0.39 nm for TIP3P and TIP4P, respectively, which characterizes the thickness of the diffusive hydration layer on the protein surface. The simulated hydration layer of CALB resulted in agreement with those experimentally determined for other seven different proteins of comparable size. By avoiding the most common pitfalls, protein diffusion can be reliably simulated: simulating different box sizes to account for the finite size effect, equilibrating the protein–water system sufficiently, and using the complete production run for the determination of the diffusion coefficient.     

Does a light-harvesting protochlorophyllide a/b-binding protein complex exist?

Recent in vitro studies have led to speculation that a novel light-harvesting protochlorophyllide a/b-binding protein complex (LHPP) might exist in dark-grown angiosperms. Structurally, it has been suggested that LHPP consists of a 5:1 ratio of dark-stable ternary complexes of the light-dependent NADPH: protochlorophyllide oxidoreductases A and B containing nonphotoactive protochlorophyllide b and photoactive protochlorophyllide a, respectively. Functionally, LHPP has been hypothesized to play major roles in establishing the photosynthetic apparatus, in protecting against photo-oxidative damage during greening, and in determining etioplast inner membrane architecture. However, the LHPP model is not compatible with other studies of the pigments and the pigment-protein complexes of dark-grown angiosperms. Protochlorophyllide b, which is postulated to be the major light-harvesting pigment of LHPP, has, for example, never been detected in etiolated seedlings. This raises the question: does LHPP exist?     

Is prostate-specific membrane antigen a multifunctional protein?

Prostate-specific membrane antigen (PSMA) is a metallopeptidase expressed predominantly in prostate cancer (PCa) cells. PSMA is considered a biomarker for PCa and is under intense investigation for use as an imaging and therapeutic target. Although the clinical utility of PSMA in the detection and treatment of PCa is evident and is being pursued, very little is known about its basic biological function in PCa cells. The purpose of this review is to highlight the possibility that PSMA might be a multifunctional protein. We suggest that PSMA may function as a receptor internalizing a putative ligand, an enzyme playing a role in nutrient uptake, and a peptidase involved in signal transduction in prostate epithelial cells. Insights into the possible functions of PSMA should improve the diagnostic and therapeutic values of this clinically important molecule. prostate cancer; receptor; peptidase; endocytosis     

Is apolipoprotein D a mammalian bilin-binding protein?

Human apolipoprotein D (APO-D) is a serum glycoprotein that has no sequence similarity with other apolipoproteins but rather belongs to the alpha 2-microglobulin superfamily whose other members transport small hydrophobic ligands in a wide variety of biological contexts. To investigate the ligand specificity of APO-D, we analyzed its relationship with the other members of this superfamily and constructed a detailed molecular model using the atomic coordinates of its most closely related homolog--insecticyanin from the tobacco hornworm, Manduca sexta. We studied the geometry of the binding pocket of APO-D and the topology of characteristic patches of both hydrophobic and polar side chains that also occur in crystal structures of insecticyanin and bilin-binding protein from the butterfly Pieris brassicae. From the data obtained we hypothesize that heme-related compounds may be more favorable ligands for APO-D than either cholesterol or cholesteryl ester. Preliminary experiments showed that purified human APO-D binds bilirubin in an approximately one-to-one molar ratio. These results suggest a new biological role for APO-D that is more congruent with its tissue distribution and evolutionary history.     

How random is a highly denatured protein?

There has been renewed interest in determining the physicochemical properties of denatured states of proteins. In many denatured states there is evidence for the existence of nonrandom configurational distributions. Here we examine the small-angle neutron scattering profile of yeast phosphoglycerate kinase in the native state and in highly denaturing conditions. We show that the denatured protein scattering profile can be interpreted using a model developed for synthetic polymers in which the chain behaves as a random coil in a good solvent, i.e. with excluded volume interactions. The implications of this result for our appreciation of the protein folding process are discussed.