MINOS (Macromolecular INsights Optimized by Scattering)

MINOS is a High-Throughput (HT) approach to structurally characterize human and human-related PSI:Biology protein targets. MINOS provides comprehensive expertise in the areas of developing and employing Small Angle X-ray Scattering (SAXS) in combination with homology modeling and high-resolution macromolecular X-ray crystallography (MX) to define accurate conformations and assemblies in solution.

Estimates suggest 40% of human proteins contain at least one intrinsically disordered region which implies nearly half of the human proteome may be intractable to traditional X-ray and NMR structure determination1,2. As a case in point, the full structural description of the p53 tumor suppressor protein has only been accomplished through the application of a comprehensive approach utilizing NMR, X-ray crystallography and SAXS. It is likely that many more protein complexes will require a comprehensive approach. Defects in transcriptional regulation, RNA splicing and modification are associated with many human diseases with several proteins such as XP-B and XP-D of the TFIIH transcription factor displaying mutation specific diseases or pleiotropy3. These proteins are often flexible in the absence of their respective binding partners or ligands and particularly for nucleic acid binding proteins, stabilization of the protein can only be achieved in complex with the requisite DNA or RNA substrate. Within the human proteome, the thousands of different transcription factors and proteins involved in RNA splicing and DNA repair and replication pose serious challenges to current high-throughput methods.

SAXS is a proven technology for providing low resolution structural information. However, SAXS is a much more powerful technique for characterizing the solution state of the biological particle. Our recent theoretical developments in SAXS analysis have established quantitative metrics for assessing particle flexibility, mass, and density (akin to the Matthews coefficient for crystallography)4,5. The scattering expertise and technologies of the SIBYLS beamline are ideally suited to achieving the goals of PSI:Biology. Our efforts have been focused on designated keystone human complexes of fundamental biological importance. MINOS fosters cohesive and mutually beneficial interactions with PSI:Biology by developing and providing innovative HT SAXS screening technologies and by contributing solution structure, conformation, and assembly results that complement PSI structures through the following aims:

Aim 1. Develop & apply SAXS methods for key biology targets.

a. Apply SAXS methods to solution structure determination of protein targets from the research community that have resisted crystallization.
b. Based upon our results and those from the research community, we will develop innovative SAXS methods, facilities, & for others.

Aim 2. Use solution scattering technologies to link structures to biology.

a. Develop & apply SAXS tools to increase our knowledge of solution shape, conformation, & complexes.
b. Increase NIH community successes by combining high-resolution structures with solution shape & flexibility data to provide informative links to biology.

References

  1. Chouard T. Structural biology: Breaking the protein rules. Nature. 2011 Mar 10;:151-3.
  2. Uversky VN, Dunker AK. Understanding protein non-folding. Biochim Biophys Acta. 2010 Jun;1804(6):1231-64.
  3. Villard J. Transcription regulation and human diseases. Swiss Med Wkly. 2004 Oct 2;134(39-40):571-9.
  4. Rambo RP, Tainer JA. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers. 2011 Aug;95(8):559-71.
  5. Rambo RP, Tainer JA. Accurate assessment of mass, models and resolution by small-angle scattering. Nature. 2013 Apr 25;496(7446):477-81.