Related ArticlesTumor suppressor p16INK4A: structural characterization of wild-type and mutant proteins by NMR and circular dichroism.
Biochemistry. 1996 Jul 23;35(29):9475-87
Authors: Tevelev A, Byeon IJ, Selby T, Ericson K, Kim HJ, Kraynov V, Tsai MD
The tumor suppressor p16INK4A with eight N-terminal amino acids deleted (p16/delta 1-8) was expressed in Escherichia coli without any fusion artifacts and purified. The integrity of p16/delta 1-8 was confirmed by mass spectrometry, and its activity was demonstrated by in vitro cdk4 inhibition assay. Various physical methods were used to characterize the molecular and structural properties of p16/delta 1-8. The protein was found to oligomerize in vitro, as demonstrated by gel electrophoresis, mass spectrometry, and NMR. Various approaches, including changes of concentration and pH, additions of salts, detergents, and various organic solvents, and construction of a C-terminal deletion mutant and a cysteine mutant were used to try to reduce the extent of oligomerization. Only decreasing the protein concentration was found to reduce oligomerization. The affinity between p16 molecules in vivo was demonstrated by the yeast two-hybrid system. The protein was found to be very unstable on the basis of urea- and guanidinium chloride-induced denaturation studies monitored by NMR and CD, respectively. Despite these unfavorable properties, total NMR assignments were accomplished with uniform 13C and 15N isotope labeling. All multidimensional NMR experiments were performed at a very low concentration of 0.2 mM. The secondary structure was then determined from the NMR data. The results of NMR and CD studies indicate that the protein is highly alpha-helical, and the ankyrin repeat sequences show helix-turn-helix structures. This is the first structural information obtained for the important motif of ankyrin repeats. Overall, p16/delta 1-8 appears to be conformationally flexible. In order to understand the structural basis of the functional changes for some mutants existing in tumor cells, several missense mutants of p16/delta 1-8 were constructed. Four of them were expressed at high levels and purified. The molecular and structural properties of these mutants were analyzed by CD and NMR and compared with the corresponding properties of wild-type p16/delta 1-8. The results suggest that the functional changes in P114L and G101W are likely to be related to global conformational changes. In addition, we have demonstrated that the tendency of aggregation increases significantly by a single D84H mutation.
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR
Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR
Abstract Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1â?˛ and C5â?˛ with...
nmrlearner
Journal club
0
11-30-2011 10:45 PM
Biochemists reveal interaction between tumor suppressor protein and chaperone - News-Medical.net
Biochemists reveal interaction between tumor suppressor protein and chaperone - News-Medical.net
<img alt="" height="1" width="1" />
Biochemists reveal interaction between tumor suppressor protein and chaperone
News-Medical.net
Using nuclear magnetic resonance (NMR) spectroscopy, the scientists at the Bavarian NMR Center in Garching were able for the first time to characterize the interaction surfaces between Hsp90 and p53 and show that p53 binds to Hsp90 in an already ...
Read here
nmrlearner
Online News
0
09-08-2011 08:24 AM
Investigation of solvent effect and NMR shielding tensors of p53 tumor-suppressor gene in drug design.
Investigation of solvent effect and NMR shielding tensors of p53 tumor-suppressor gene in drug design.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Investigation of solvent effect and NMR shielding tensors of p53 tumor-suppressor gene in drug design.
Int J Nanomedicine. 2011;6:213-8
Authors: Irani S, Monajjemi M, Honarparvar B, Atyabi S, Sadeghizadeh M
Abstract
The p53 tumor-suppressor gene encodes a nuclear phosphoprotein with cancer- inhibiting properties. The...
nmrlearner
Journal club
0
08-25-2011 04:10 PM
An NMR study of the N-terminal domain of wild-type hERG and a T65P trafficking deficient hERG mutant.
An NMR study of the N-terminal domain of wild-type hERG and a T65P trafficking deficient hERG mutant.
An NMR study of the N-terminal domain of wild-type hERG and a T65P trafficking deficient hERG mutant.
Proteins. 2011 May 16;
Authors: Gayen S, Li Q, Chen AS, Nguyen TH, Huang Q, Hill J, Kang C
The human Ether-ŕ-go-go Related Gene (hERG) potassium channel plays an important role in the heart by controlling the rapid delayed rectifier current. The N-terminal 135 residues (NTD) contain a Per-Arnt-Sim (PAS) domain and an N-terminal amphipathic helix....
nmrlearner
Journal club
0
06-12-2011 12:15 AM
NMR Characterization of a “Fibril-Ready” State of Demetalated Wild-Type Superoxide Dismutase
NMR Characterization of a “Fibril-Ready” State of Demetalated Wild-Type Superoxide Dismutase
Lucia Banci, Ivano Bertini, Olga Blaževitš, Francesca Cantini, Moreno Lelli, Claudio Luchinat, Jiafei Mao and Miguela Vieru
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja1069689/aop/images/medium/ja-2010-069689_0006.gif
Journal of the American Chemical Society
DOI: 10.1021/ja1069689
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/uAjKy7vWoHs
nmrlearner
Journal club
0
12-17-2010 12:50 AM
[NMR paper] Tumor suppressor INK4: refinement of p16INK4A structure and determination of p15INK4B
Tumor suppressor INK4: refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data.
Related Articles Tumor suppressor INK4: refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data.
Protein Sci. 2000 Jun;9(6):1120-8
Authors: Yuan C, Selby TL, Li J, Byeon IJ, Tsai MD
Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined...
nmrlearner
Journal club
0
11-18-2010 09:15 PM
[NMR paper] Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif Related Articles Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Eur J Biochem. 1991 Aug 15;200(1):139-48
Authors: Folkers PJ, Stassen AP, van Duynhoven JP, Harmsen BJ, Konings RN, Hilbers CW
Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the...
nmrlearner
Journal club
0
08-21-2010 11:12 PM
[NMR paper] Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif Related Articles Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Eur J Biochem. 1991 Aug 15;200(1):139-48
Authors: Folkers PJ, Stassen AP, van Duynhoven JP, Harmsen BJ, Konings RN, Hilbers CW
Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the...