Structural characterization of DNA-binding domain of essential mammalian protein TTF 1
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Abstract
Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues). Despite its critical role in various cellular processes, the physical structure of TTF1 remains unsolved. Attempts to purify the functional TTF1 protein have been unsuccessful till date. Therefore, we focused on characterizing the Myb domain of this essential protein. We started with predicting a 3-D model of the Myb domain using homology modelling, and ab-initio method. We then determined its stability through MD simulation in an explicit solvent. The model predicted is highly stable, which stabilizes at 200ns. To experimentally validate the computational model, we cloned and expressed the codon optimized Myb domain into a bacterial expression vector and purified the protein to homogeneity. Further, characterization of the protein shows that, Myb domain is predominantly helical (65%) and is alone sufficient to bind the Sal Box DNA. This is the first-ever study to report a complete in silico model of the Myb domain, which is physically characterized. The above study will pave the way towards solving the atomic structure of this essential mammalian protein.
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The results validated the computational model, concluding that this domain is predominantly helical in nature. The confidence built by this study now pushes us to move ahead in order to solve the atomic structure of this critical domain by crystallography or NMR spectroscopy, which in turn will decipher the exact mechanism by which this essential protein engages DNA to cater to various functions
What a cool paper combining computational modeling of protein structure with experimental analysis to support it! I'm excited for the next steps listed here (crystallography or NMR) to see how those results line up with what was found here, but either way, I'm a big fan of the combined computational and experimental work!
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we used Raman spectroscopy. Raman spectrum of the buffer (control) is displayed in black, whereas that of the Myb domain is displayed in red
Did you do technical replicates? I'm curious about how consistent the data is between trials.
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Graphical quantification of intensity of the protein-DNA complex formed
I'm curious if you happened to do replicates of this and/or stats to determine if your quantification is significant?
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AlphaFold, SWISS-MODEL, and Robetta predicted compact and ordered structures with a very high percentage of α-helical conformations. While the model predicted by I-TASSER was less compact and ordered compared to the models generated by the above servers. All models have very similar and reliable statistics as per the overall SAVESv6.0 results. In summary, the structural integrity and statistics of the models derived from both homology and ab-initio methods showed considerable consistency. This confirms the reliability of Robetta and other models (excluding I-TASSER) for further computational analysis.
I really appreciate the direct comparison of the 4 methods on a single sample. This is totally beyond the scope of the paper, but I wonder if these observations would hold true with other proteins/protein domains.
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27%
Is this not just because you're BLASTing a single domain against a database of mostly full proteins?
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protein
It's a bit confusing to refer to the Myb domain as a "protein" when you aren't talking about the full-length protein.
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(Accession Number: Q62187)
Love that you included the accession number and a direct link to the sequence! It can be a pain when papers don't reference the exact protein that they're working with. It might be worth explicitly stating how you identified the Myb domain.
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neither an in silico nor a physically determined structure of the individual domains of TTF1 is available to date
Just curious if there's a structure available of the full protein? It might be useful context to see how your analysis of the Myb domain fits with the full structure (if it's available).
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DDB1
Do you know the domains that these different interacting proteins bind? The first sentence suggests that you do, and it might be useful for better understanding the particular domain that you focus on here (Myb) to know what interacts with it.
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ranging from 323 to 445 amino acids
This is very minor, but when I first read this (before looking at Figure 1), I thought this phrase meant that this specific region was 323-445 amino acids long. It might be less confusing to use language like "a specific region located between amino acid 323 and 445"? Otherwise, this section introducing the functional domains is very thorough and got me right up to speed on TTF1!
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