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PubMed1996

Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO.

The gene (ssb) encoding the single-stranded DNA binding (SSB) protein from Pseudomonas aeruginosa PAO was detected on a 2.1 kbp PstI-fragment of chromosomal DNA. The protein (PaeSSB) encoded by this gene consists of 165 aa and has a M(r) of 18549. The genomic sequence was confirmed by amino acid sequencing of the amino terminus of SSB protein isolated from P. aeruginosa PAO. PaeSSB shows 68% homology to the respective protein of E. coli. The nucleotide sequence upstream of the P. aeruginosa ssb gene shows little homology to the regulatory region upstream of the ssb gene of E. coli. The ssb gene was located at a distance of 690-870 kbp from the origin of replication on a physical map of P. aeruginosa PAO. In vivo PaeSSB could replace the SSB protein of E. coli (EcoSSB) if its production was controlled by the lac promoter on a high-copy vector. PaeSSB was overproduced in E. coli. Both the overproduced protein and PaeSSB isolated from Pseudomonas aeruginosa PAO are post-translationally modified by cleavage of the first methionine. Analytical ultracentrifugation shows that PaeSSB is a stable homotetramer. The copy number of PaeSSB in P. aeruginosa is 1200 +/- 250 tetramers per cell. Preliminary characterization of the DNA binding properties shows PaeSSB to have a lower affinity for single-stranded DNA than EcoSSB.

Amino Acid Sequence↗

PubMed1990

Stabilization of recA protein-ssDNA complexes by the single-stranded DNA binding protein of Escherichia coli.

In vitro recombination reactions promoted by the recA protein of Escherichia coli are enhanced by the single-stranded DNA binding protein (SSB). SSB affects the assembly of the filamentous complexes between recA protein and ssDNA that are the active form of the recA protein. Here, we present evidence that SSB plays a complex role in maintaining the stability and activity of recA-ssDNA filaments. Results of ATPase, nuclease protection, and DNA strand exchange assays suggest that the continuous presence of SSB is required to maintain the stability of recA-ssDNA complexes under reaction conditions that support their recombination activity. We also report data that indicate that there is a functional distinction between the species of SSB present at 10 mM magnesium chloride, which enhances recA-ssDNA binding, and a species present at 1 mM magnesium chloride, which displaces recA protein from ssDNA. These results are discussed in the context of current models of SSB conformation and of SSB action in recombination activities of the recA protein.

Adenosine Triphosphate↗

PubMed1990

Kinetics of binding of single-stranded DNA binding protein from Escherichia coli to single-stranded nuclei acids.

The time course of the reaction of Escherichia coli single-stranded DNA binding protein (E. coli SSB) with poly(dT) and M13mp8 single-stranded DNA has been measured by fluorescence stopped-flow experiments. For poly(dT), the fluorescence traces follow simple bimolecular behavior up to 80% saturation of the polymer with E. coli SSB. A mechanistic explanation of this binding behavior can be given as follows: (1) E. coli SSB is able to translocate very rapidly on the polymer, forming cooperative clusters. (2) In the rate-limiting step of the association reaction, E. coli SSB is bound to the polymer only by one or two of its four contact sites. As compared to poly(dT), association to single-stranded M13mp8 phage DNA is slower by at least 2 orders of magnitude. We attribute this finding to the presence of secondary structure elements (double-stranded structures) in the natural single-stranded DNA. These structures cannot be broken by E. coli SSB in a fast reaction. In order to fulfill its physiological function in reasonable time, E. coli SSB must bind newly formed single-stranded DNA immediately. The protein can, however, bind to such pieces of the newly formed single-stranded DNA which are too short to cover all four binding sites of the E. coli SSB tetramer.

Coliphages↗

PubMed1981

Escherichia coli single-strand deoxyribonucleic acid binding protein: stability, specificity, and kinetics of complexes with oligonucleotides and deoxyribonucleic acid.

The complex formation between the single-strand DNA binding protein (ssB protein) from Escherichia coli and oligonucleotides and single-stranded DNA has been studied by using fluorescence titrations, ultracentrifugation measurements, and fast kinetic techniques. Determination of the stoichiometries of oligo(dT)--ssB complexes shows that each of the four subunits of the ssB protein represents a binding site for an oligonucleotide about eight residues long. Occupation of all four binding sites with oligo(dT) or poly(dT) leads to 80% quenching of the intrinsic protein fluorescence. The binding sites are nearly equivalent and independent. For d(pT)16, the intrinsic binding constant is 6 X 10(5) M-1, and for d(pT)30-40, which is long enough to extend continuously over the ssB tetramer, the binding constant is higher than 5 X 10(8) M-1. Oligoadenylates bind about 2 orders of magnitude weaker than the corresponding oligo(dT) species. The binding of oligo(dT) is very weakly dependent on ionic strength, in contrast to the oligo(dA)--ssB complex formation. For d(pT)8, d(pT)16, and d(pT)30-40, the complex formation can be described by a simple one-step reaction. The strength of the interaction is mainly expressed in the rate constant of dissociation. In the cooperative complexes with poly(dT) or poly(dA), all four binding sites on the ssB tetramer are also occupied. It is concluded that single-stranded DNA is coiled around the ssB molecule. Fluorescence melting experiments of the complexes show that the conformation of the single-stranded DNA has a strong influence on the stability of the complexes.

Binding Sites↗

PubMed1999

A DNA probe specific to Streptococcus sobrinus.

Three DNA fragments (SSB-1, -2 and -3) in the dextranase gene (dex) of Streptococcus sobrinus were amplified by polymerase chain reaction and used as DNA probes. The probes were examined for the specificity and the sensitivity of hybridization with DNA of oral streptococcal species. While probes SSB-1 and SSB-2 were specific to both S. sobrinus and Streptococcus downei, SSB-3 was specific only to S. sobrinus. SSB-3 was able to detect 5 ng of chromosomal DNA purified from S. sobrinus NIDR6715 and DNA extracted from 1 x 10(5) cells of the strain. In addition, SSB-3 could differentiate clinical isolates of S. sobrinus from Streptococcus mutans. These results suggest that SSB-3 is an effective DNA-probe to detect and to identify S. sobrinus.

Bacterial Proteins↗

PubMed1982

Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units.

Electron microscopy shows that complexes of the single-strand DNA binding protein (SSB) of Escherichia coli and phage fd DNA appear as beaded fiber loops containing an average of 38 beads, 1 per 170 bases of DNA. Extensive digestion of native unfixed SSB-fd DNA complexes with micrococcal nuclease reveals a protected DNA fragment of 145 bases, while shorter digestion periods result in a sequence of fragments in multiples of 160 +/- 25 bases. Digestion of these complexes with DNase I produces a repeating pattern of bands, multiples of approximately 15 bases with strong bands at 60, 105, 118, 130, 145, 150, and 210 bases. Isopycnic banding in CsCl solution yields densities of 1.272 and 1.700 g/ml, respectively, for SSB alone and for fd DNA and, after fixation, of 1.388 g/ml for fd DNA-SSB beaded fibers and 1.373 g/ml for the individual protein-DNA beads. Based on these data and the molecular weights of SSB and fd DNA, we suggest that the nucleoprotein chain consists of eight molecules of SSB bound to 145 bases of DNA, with these units linked by roughly 30 bases of protein-free DNA. The excellent concord between results obtained by enzyme digestion of unfixed native samples and, after fixation, by electron microscopy and density banding supports the conclusion that SSB organizes single-stranded DNA in a manner similar to the organization of duplex DNA by histones.

Carrier Proteins↗

PubMed1986

Isolation of the gene encoding yeast single-stranded nucleic acid binding protein 1.

A yeast gene encoding SSB-1, a single-stranded nucleic acid binding protein, has been isolated by screening a lambda gt11 genomic DNA library. The gene is located on a 1.84-kilobase chromosomal Bgl II-BamHI fragment. Yeast strains carrying the high-copy-number vector YEp24 with an SSB1 gene insert overproduce SSB-1 3-fold and SSB-1 mRNA 10-fold. A typical haploid cell contains about 20,000 molecules of SSB-1; thus, the cells can tolerate up to 60,000 copies. Yeast SSB-1 was expressed in Escherichia coli cells by using a phage T7 expression system. Spores containing the gene disrupted at a point within the coding sequence germinate and grow normally; thus, the gene is not essential. Protein blots show that no SSB-1 or novel immunologically related species that might retain SSB-1 activity are present in cells containing the disrupted SSB1 genes. Southern analysis and protein blots suggest the presence in yeast of a second, related, but nonidentical gene and two immunologically related proteins of 55 kDa and 75 kDa.

DNA, Recombinant↗

PubMed1993

Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein.

The Escherichia coli RecF, RecO, and RecR proteins were analyzed for their effect on RecA-mediated pairing of single-stranded circular DNA and homologous linear duplex DNA substrates. As shown by other workers, joint molecule formation by RecA was inhibited by E. coli single-stranded DNA binding protein (SSB) when it was added to single-stranded DNA before RecA. This inhibitory effect was overcome by the addition of RecO and RecR or RecF, RecO, and RecR. Both the rate and extent of joint molecule formation were restored to the maximal level observed when SSB was added after RecA. RecF, RecO, and RecR proteins had no effect on the conversion of joint molecules to final products and only appeared to stimulate an early step in the pairing reaction. The stimulatory effect of RecF, RecO, and RecR was not seen without SSB or when SSB was added after RecA. RecF protein by itself inhibited reactions in mixtures containing RecA and SSB, and this inhibition was overcome by the addition of RecO and RecR. These data suggest that RecO and RecR, and possibly RecF, help RecA overcome inhibition by SSB and utilize SSB-single-stranded-DNA complexes as substrates.

Bacterial Proteins↗

PubMed1998

The biochemical properties of the ATPase activity of a 70-kDa heat shock protein (Hsp70) are governed by the C-terminal domains.

The cytosolic 70-kDa heat shock proteins (Hsp70s), Ssa and Ssb, of Saccharomyces cerevisiae are functionally distinct. Here we report that the ATPase activities of these two classes of Hsp70s exhibit different kinetic properties. The Ssa ATPase has properties similar to those of other Hsp70s studied, such as DnaK and Hsc70. Ssb, however, has an unusually low steady-state affinity for ATP but a higher maximal velocity. In addition, the ATPase activity of Hsp70s, like that of Ssa1, depends on the addition of K+ whereas Ssb activity does not. Suprisingly, the isolated 44-kDa ATPase domain of Ssb has a Km and Vmax for ATP hydrolysis similar to those of Ssa, rather than those of full length Ssb. Analysis of Ssa/Ssb fusion proteins demonstrates that the Ssb peptide-binding domain fused to the Ssa ATPase domain generates an ATPase of relatively high activity and low steady-state affinity for ATP similar to that of native Ssb. Therefore, at least some of the biochemical differences between the ATPases of these two classes of Hsp70s are not intrinsic to the ATPase domain itself. The differential influence of the peptide-binding domain on the ATPase domain may, in part, explain the functional uniqueness of these two classes of Hsp70s.

Adenosine Triphosphatases↗

PubMed2002

Importance of the N-terminal region of the phage GA-1 single-stranded DNA-binding protein for its self-interaction ability and functionality.

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages phi 29 and Nf. In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of phi 29 and Nf. Three deletion mutant proteins have been characterized, Delta N19, Delta N26, and Delta N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein Delta N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins Delta N26 and Delta N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.

Amino Acid Sequence↗

PubMed2003

The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein.

The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions. The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein. The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein. Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A. The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.

Adenosine Triphosphate↗

PubMed2005

Hsp110 cooperates with different cytosolic HSP70 systems in a pathway for de novo folding.

Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bind mostly post-translationally to a more restricted subset of newly translated polypeptides, suggesting a downstream function in the folding pathway. Notably, loss of SSB dramatically enhances the cotranslational association of SSA with nascent chains, suggesting SSA can partially fulfill an SSB-like function. On the other hand, the absence of SSE1 enhances polypeptide binding to both SSB and SSA and impairs cell growth. It, thus, appears that Hsp110 is an important regulator of Hsp70-substrate interactions. Based on our data, we propose that Hsp110 cooperates with the SSB and SSA Hsp70 subfamilies, which act sequentially during de novo folding.

Adenosine Triphosphate↗

PubMed2005

Effect of Mg2+ on the DNA binding modes of the Streptococcus pneumoniae SsbA and SsbB proteins.

The effect of Mg2+ on the binding of the Streptococcus pneumoniae single-stranded DNA binding (SSB) proteins, SsbA and SsbB, to various dT(n) oligomers was examined by polyacrylamide gel electrophoresis. The results were then compared with those that were obtained with the well characterized SSB protein from Escherichia coli, SsbEc. In the absence of Mg2+, the results indicated that the SsbEc protein was able to bind to the dT(n) oligomers in the SSB(35) mode, with only two of the four subunits of the tetramer interacting with the dT(n) oligomers. In the presence of Mg2+, however, the results indicated that the SsbEc protein was bound to the dT(n) oligomers in the SSB(65) mode, with all four subunits of the tetramer interacting with the dT(n) oligomers. The SsbA protein behaved similarly to the SsbEc protein under all conditions, indicating that it undergoes Mg2+ -dependent changes in its DNA binding modes that are analogous to those of the SsbEc protein. The SsbB protein, in contrast, appeared to bind to the dT(n) oligomers in an SSB(65)-like mode in either the presence or the absence of Mg2+, suggesting that it may not exhibit the pronounced negative intrasubunit cooperativity in the absence of Mg2+ that is required for the formation of the SSB(35) mode. Additional experiments with a chimeric SsbA/B protein indicated that the structural determinants that govern the transitions between the different DNA binding modes may be contained within the N-terminal domains of the SSB proteins.

Amino Acid Sequence↗

PubMed2003

Occupational exposure to heavy metals: DNA damage induction and DNA repair inhibition prove co-exposures to cadmium, cobalt and lead as more dangerous than hitherto expected.

Co-exposure to cadmium, cobalt, lead and other heavy metals occurs in many occupational settings, such as pigment and batteries production, galvanization and recycling of electric tools. However, little is known about interactions between several heavy metals. In the present study we determined DNA single strand break (DNA-SSB) induction and repair capacity for 8-oxoguanine in mononuclear blood cells of 78 individuals co-exposed to cadmium (range of concentrations in air: 0.05-138.00 micro g/m(3)), cobalt (range: 0-10 micro g/m(3)) and lead (range: 0-125 micro g/m(3)). Exposure to heavy metals was determined in air, blood and urine. Non-parametric correlation analysis showed a correlation between cadmium concentrations in air with DNA-SSB (P = 0.001, R = 0.371). Surprisingly, cobalt air concentrations correlated even better (P < 0.001, R = 0.401), whereas lead did not correlate with DNA-SSB. Logistic regression analysis including 11 possible parameters of influence resulted in a model showing that cobalt in air, cadmium in air, cadmium in blood and lead in blood influence the level of DNA-SSB. The positive result with cobalt was surprising, since exposure levels were much lower compared with the TRK-value of 100 micro g/m(3). To examine, whether the positive result with cobalt is stable, we applied several logistic regression models with two blocks, where all factors except cobalt were considered preferentially. All strategies resulted in the model described above. Logistic regression analysis considering also all possible interactions between the relevant parameters of influence finally resulted in the following model: Odds ratio = 1.286(Co in air) x 1.040(Cd in air) x 3.111(Cd in blood) x 0.861(Pb in air) x 1.023(Co in air x Pb in air). This model correctly predicts an increased level of DNA-SSB in 91% of the subjects in our study. One conclusion from this model is the existence of more than multiplicative effects for co-exposures of cadmium, cobalt and lead. For instance increasing lead air concentrations from 1.6 to 50 micro g/m(3) in the presence of constant exposures to cobalt and cadmium (8 micro g/m(3) and 3.8 micro g/m(3)) leads to an almost 5-fold increase in the odds ratio, although lead alone does not increase DNA-SSB. The mechanism behind these interactions might be repair inhibition of oxidative DNA damage, since a decrease in repair capacity will increase susceptibility to reactive oxygen species generated by cadmium or cobalt. Indeed, repair of 8-oxoguanine decreased with increasing exposures and inversely correlated with the level of DNA-SSB (P = 0.001, R = -0.427). Protein expression patterns of individuals exposed to cobalt concentrations of approximately 10 micro g/m(3) were compared with those of unexposed individuals using two-dimensional gel electrophoresis. Qualitative and apparent quantitative alterations in protein expression were selective and certainly occurred in <0.1% of all proteins. In conclusion, the hazard due to cobalt exposure - that has been classified only as IIB by the IARC - seems to be underestimated, especially when individuals are co-exposed to cadmium or lead. Co-exposure may cause genotoxic effects, even if the concentrations of individual heavy metals do not exceed TRK-values.

Alcohol Drinking↗

PubMed1990

Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure.

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.

Chromatography, Gel↗

PubMed1999

Differential effects of single-stranded DNA binding proteins (SSBs) on uracil DNA glycosylases (UDGs) from Escherichia coli and mycobacteria.

Deamination of cytosines results in accumulation of uracil residues in DNA, which unless repaired lead to GC-->AT transition mutations. Uracil DNA glyco-sylase excises uracil residues from DNA and initiates the base excision repair pathway to safeguard the genomic integrity. In this study, we have investigated the effect of single-stranded DNA binding proteins (SSBs) from Escherichia coli (Eco SSB) and Mycobacterium tuberculosis (Mtu SSB) on uracil excision from synthetic substrates by uracil DNA glycosylases (UDGs) from E. coli, Mycobacterium smegmatis and M.tuberculosis (referred to as Eco -, Msm - and Mtu UDGs respectively). Presence of SSBs with all the three UDGs resulted in decreased efficiency of uracil excision from a single-stranded 'unstructured' oligonucleo-tide, SS-U9. On the other hand, addition of Eco SSB to Eco UDG, or Mtu SSB to Mtu UDG reactions resulted in increased efficiency of uracil excision from a hairpin oligonucleotide containing dU at the second position in a tetraloop (Loop-U2). Interestingly, the efficiency of uracil excision by Msm UDG from the same substrate was decreased in the presence of either Eco- or Mtu SSBs. Furthermore, Mtu SSB also decreased uracil excision from Loop-U2 by Eco UDG. Our studies using surface plasmon resonance technique demonstrated interactions between the homologous combinations of SSBs and UDGs. Heterologous combinations either did not show detectable interaction (Eco SSB with Mtu UDG) or showed a relatively weaker interaction (Mtu SSB with Eco UDG). Taken together, our studies suggest differential interactions between the two groups (SSBs and UDGs) of the highly conserved proteins. Such studies may provide important clues to design selective inhibitors against this important class of DNA repair enzymes.

DNA↗

PubMed1992

Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection. The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs. In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured. We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription. We report here that Escherichia coli single-stranded DNA-binding protein (SSB) is also required for N4 early transcription. In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription. The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation. The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute. Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription. The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.

Base Sequence↗

PubMed2006

Dynamics of the SPRY domain-containing SOCS box protein 2: flexibility of key functional loops.

The SPRY domain was identified originally as a sequence repeat in the dual-specificity kinase splA and ryanodine receptors and subsequently found in many other distinct proteins, including more than 70 encoded in the human genome. It is a subdomain of the B30.2/SPRY domain and is believed to function as a protein-protein interaction module. Three-dimensional structures of several B30.2/SPRY domain-containing proteins have been reported recently: murine SSB-2 in solution by NMR spectroscopy, a Drosophila SSB (GUSTAVUS), and human PRYSPRY protein by X-ray crystallography. The three structures share a core of two antiparallel beta-sheets for the B30.2/SPRY domain but show differences located mainly at one end of the beta-sandwich. Analysis of SSB-2 residues required for interactions with its intracellular ligands has provided insights into B30.2/SPRY binding specificity and identified loop residues critical for the function of this domain. We have investigated the backbone dynamics of SSB-2 by means of Modelfree analysis of its backbone (15)N relaxation parameters and carried out coarse-grained dynamics simulation of B30.2/SPRY domain-containing proteins using normal mode analysis. Translational self-diffusion coefficients of SSB-2 measured using pulsed field gradient NMR were used to confirm the monomeric state of SSB-2 in solution. These results, together with previously reported amide exchange data, highlight the underlying flexibility of the loop regions of B30.2/SPRY domain-containing proteins that have been shown to be important for protein-protein interactions. The underlying flexibility of certain regions of the B30.2/SPRY domain-containing proteins may also contribute to some apparent structural differences observed between GUSTAVUS or PRYSPRY and SSB-2.

Amino Acid Sequence↗