X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase

Polyakov, K. M.; Goncharuk, D. A.; Trofimov, A.A.; Safonova, T. N.; Mitkevich, Vladimir A.; Tkach, Elena N.; Макаров, А. А.; Shulga, A. A.

doi:10.1134/s0026893310050195

Cited by 6 publications

(7 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have shown that binase exists on SDS-PAGE in both its monomeric and dimeric forms ( Fig. 1A ), which fits with previous data showing that binase forms dimers in a crystal and probably in solutions with high protein concentrations [8] , [15] . The question is addressed to the ratio of binase monomers and dimers in solution in the absence of denaturing agents.…”

Section: Resultssupporting

confidence: 91%

“…In the crystal structures of binase, the characteristic dimer is present with the active site of one subunit being blocked owing to interactions within the dimer [8] . Since the dimers were found in crystals grown under four different conditions, it can be suggested that the enzyme exists as a dimer in solution as well [15] . Although only one native dimeric RNase, the bull semen RNase (BS-RNase), has been identified to date [16] , RNase A can also form dimers under certain conditions [17] .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

et al. 2014

View full text Add to dashboard Cite

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.

show abstract

Section: Resultssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Within this family, three-dimensional structures of ribonucleases from bacteria, actinomycetes and fungi have been determined (Heinemann et al, 2003). Previously, the following three-dimensional structures of binase have been determined: the free enzyme at 1.65 Å resolution, the enzyme in complex with sulfate at 2.0 Å resolution, the enzyme in complex with guanosine 3 0 -phosphate at 2.0 Å resolution (Polyakov et al, 2002) and two structures of the Trp34Phe mutant at 1.1 and 1.7 Å resolution (Polyakov et al, 2010). In all of the crystal structures determined previously, binase exists in a dimeric form.…”

Section: Introductionmentioning

confidence: 99%

Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant

Mitkevich

Schulga

Trofimov

et al. 2013

Acta Crystallogr D Biol Cryst

Self Cite

View full text Add to dashboard Cite

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

show abstract

“…Existence of two molecular forms of binase, a monomeric and a dimeric one, was considered by Polyakov et al [34], Konovalova et al [35], Shirshikov et al [36], and Ermakova [37]. In these works, how ever, existence of dimeric structures formed due to hydrogen bonds and hydrophobic interactions was theoretically proposed in binase crystals, while their formation in solutions was considered hypothetical [34,36].…”

Section: Resultsmentioning

confidence: 99%

“…In these works, how ever, existence of dimeric structures formed due to hydrogen bonds and hydrophobic interactions was theoretically proposed in binase crystals, while their formation in solutions was considered hypothetical [34,36]. Formation of dimeric and aggregated forms of binase on the membrane surface, which may enhance the penetration of this enzyme into the cell, is also probable [35].…”

Section: Resultsmentioning

confidence: 99%

Preparations of Bacillus pumilus secreted RNase: One enzyme or two?

et al. 2015

View full text Add to dashboard Cite

Immunochemical analysis of the following purified preparations of Bacillus pumilus RNase (binase) was carried out: industrially manufactured enzyme (Institute of Organic Synthesis, Riga, Latvia) and the enzymes isolated from the culture liquid of the native B. pumilus producer and from the Escherichia coli BL21 recombinant strain bearing the pGEMGX1/ent/Bi plasmid. Electrophoresis of all three samples of purified binase revealed two protein fractions with ribonuclease activity possessing molecular masses of ~12 and 25 kDa. The possible presence of binase II, a second secreted RNase, was ruled out. Both high and low molecular mass proteins interacted with binase specific antibodies in the immunoblotting reaction, which indicated their antigenic identity. The difference in molecular mass between these proteins indicated the pos sible presence of two forms of binase in solution, a monomer and a dimer.

show abstract

X-ray diffraction and biochemical studies of W34F mutant ribonuclease binase

Cited by 6 publications

References 9 publications

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant

Preparations of Bacillus pumilus secreted RNase: One enzyme or two?

Contact Info

Product

Resources

About