Limits...
Characterization of a core fragment of the rhesus monkey TRIM5α protein.

Kar AK, Mao Y, Bird G, Walensky L, Sodroski J - BMC Biochem. (2011)

Bottom Line: This BCCL2 protein formed dimers and higher-order oligomers in solution.Approximately 40% of the BCCL2 secondary structure consisted of alpha helices.These results indicate that the B-box 2, coiled-coil and linker 2 regions of TRIM5α form a core dimerization motif that exhibits a high level of alpha-helical content.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT

Background: Like all tripartite motif (TRIM) proteins, the retroviral restriction factor TRIM5α consists of RING, B-box 2 and coiled-coil domains, with a C-terminal B30.2(SPRY) domain. Although structures have been determined for some individual TRIM domains, the structure of an intact TRIM protein is unknown.

Results: Here, we express and characterize a protease-resistant 29-kD core fragment containing the B-box 2, coiled coil and adjacent linker (L2) region of TRIM5α. This BCCL2 protein formed dimers and higher-order oligomers in solution. Approximately 40% of the BCCL2 secondary structure consisted of alpha helices. Partial loss of alpha-helical content and dissociation of dimers occurred at 42°C, with the residual alpha helices remaining stable up to 80°C.

Conclusions: These results indicate that the B-box 2, coiled-coil and linker 2 regions of TRIM5α form a core dimerization motif that exhibits a high level of alpha-helical content.

Show MeSH

Related in: MedlinePlus

Size-exclusion chromatography-light scattering (SEC-LS) analysis of the LLER protein. A. Approximately 300 μg of the purified LLER protein was applied to a Superose 6 HR 10/30 column coupled with an in-line Dawn EOS laser light-scattering apparatus, refractometer and UV detector. The dashed red line represents the light-scattering signal at 90°. The green trace represents the refractive index. The blue trace represents the UV absorption at 280 nm. B. The solid red line indicates the refractometer trace of Peak 1 (P1) and Peak 2 (P2). The "dots" represent the weight-average molecular mass for each slice, measured every second. The results of the analysis performed on the protein in Peak 2 (P2) are shown beneath the figure. The table summarizes the analyses of Peaks 1 and 2.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3025952&req=5

Figure 5: Size-exclusion chromatography-light scattering (SEC-LS) analysis of the LLER protein. A. Approximately 300 μg of the purified LLER protein was applied to a Superose 6 HR 10/30 column coupled with an in-line Dawn EOS laser light-scattering apparatus, refractometer and UV detector. The dashed red line represents the light-scattering signal at 90°. The green trace represents the refractive index. The blue trace represents the UV absorption at 280 nm. B. The solid red line indicates the refractometer trace of Peak 1 (P1) and Peak 2 (P2). The "dots" represent the weight-average molecular mass for each slice, measured every second. The results of the analysis performed on the protein in Peak 2 (P2) are shown beneath the figure. The table summarizes the analyses of Peaks 1 and 2.

Mentions: The oligomeric state of the LLER protein, purified by nickel-affinity and anion-exchange chromatography, was investigated by size-exclusion chromatography-light scattering (SEC-LS). SEC-LS can estimate molecular mass independently of the Stokes radius of the protein and without reference to a curve based on protein standards. Molecular mass determination by SEC-LS depends only upon the light-scattering and refractive indices, which are measured by detectors situated downstream of the size-exclusion column. The LLER protein eluted in two peaks, the first ranging from 140-160 kD, the second from 57-61 kD (Figure 5A). A size analysis by Astra (Figure 5B) calculated a hydrodynamic radius of approximately 4.8 nm. The molecular weight of the protein in Peak 2 is consistent with that expected for a dimer, whereas the protein in Peak 1 apparently represents a higher-order oligomer.


Characterization of a core fragment of the rhesus monkey TRIM5α protein.

Kar AK, Mao Y, Bird G, Walensky L, Sodroski J - BMC Biochem. (2011)

Size-exclusion chromatography-light scattering (SEC-LS) analysis of the LLER protein. A. Approximately 300 μg of the purified LLER protein was applied to a Superose 6 HR 10/30 column coupled with an in-line Dawn EOS laser light-scattering apparatus, refractometer and UV detector. The dashed red line represents the light-scattering signal at 90°. The green trace represents the refractive index. The blue trace represents the UV absorption at 280 nm. B. The solid red line indicates the refractometer trace of Peak 1 (P1) and Peak 2 (P2). The "dots" represent the weight-average molecular mass for each slice, measured every second. The results of the analysis performed on the protein in Peak 2 (P2) are shown beneath the figure. The table summarizes the analyses of Peaks 1 and 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3025952&req=5

Figure 5: Size-exclusion chromatography-light scattering (SEC-LS) analysis of the LLER protein. A. Approximately 300 μg of the purified LLER protein was applied to a Superose 6 HR 10/30 column coupled with an in-line Dawn EOS laser light-scattering apparatus, refractometer and UV detector. The dashed red line represents the light-scattering signal at 90°. The green trace represents the refractive index. The blue trace represents the UV absorption at 280 nm. B. The solid red line indicates the refractometer trace of Peak 1 (P1) and Peak 2 (P2). The "dots" represent the weight-average molecular mass for each slice, measured every second. The results of the analysis performed on the protein in Peak 2 (P2) are shown beneath the figure. The table summarizes the analyses of Peaks 1 and 2.
Mentions: The oligomeric state of the LLER protein, purified by nickel-affinity and anion-exchange chromatography, was investigated by size-exclusion chromatography-light scattering (SEC-LS). SEC-LS can estimate molecular mass independently of the Stokes radius of the protein and without reference to a curve based on protein standards. Molecular mass determination by SEC-LS depends only upon the light-scattering and refractive indices, which are measured by detectors situated downstream of the size-exclusion column. The LLER protein eluted in two peaks, the first ranging from 140-160 kD, the second from 57-61 kD (Figure 5A). A size analysis by Astra (Figure 5B) calculated a hydrodynamic radius of approximately 4.8 nm. The molecular weight of the protein in Peak 2 is consistent with that expected for a dimer, whereas the protein in Peak 1 apparently represents a higher-order oligomer.

Bottom Line: This BCCL2 protein formed dimers and higher-order oligomers in solution.Approximately 40% of the BCCL2 secondary structure consisted of alpha helices.These results indicate that the B-box 2, coiled-coil and linker 2 regions of TRIM5α form a core dimerization motif that exhibits a high level of alpha-helical content.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT

Background: Like all tripartite motif (TRIM) proteins, the retroviral restriction factor TRIM5α consists of RING, B-box 2 and coiled-coil domains, with a C-terminal B30.2(SPRY) domain. Although structures have been determined for some individual TRIM domains, the structure of an intact TRIM protein is unknown.

Results: Here, we express and characterize a protease-resistant 29-kD core fragment containing the B-box 2, coiled coil and adjacent linker (L2) region of TRIM5α. This BCCL2 protein formed dimers and higher-order oligomers in solution. Approximately 40% of the BCCL2 secondary structure consisted of alpha helices. Partial loss of alpha-helical content and dissociation of dimers occurred at 42°C, with the residual alpha helices remaining stable up to 80°C.

Conclusions: These results indicate that the B-box 2, coiled-coil and linker 2 regions of TRIM5α form a core dimerization motif that exhibits a high level of alpha-helical content.

Show MeSH
Related in: MedlinePlus