3DS8 Secondary Structure
The discovery and characterization of the structure and function of the protein 3DS8.
This project aimed to characterize the function of an unknown protein 3DS8 with a known structure by comparing it to known proteins and subjecting it to different tests in the laboratory.
Different techniques to achieve this goal include protein expression, purification and analysis, kinetics, and computational methods.
Function
The proposed function of the unknown protein 3DS8 is hydrolase activity. It has many structural similarities to known proteins with hydrolase and protease activity.
The function was not confirmed during wet lab experiments due to protein and reagent complications.
Experimental
The beginning experiments were conducted on molecular docking sites to compare the structure of the 3DS8 active site with known proteins. Using the SPRITE database [1],
the 3DS8 protein was matched with a few proteases through right-handed superpositions with RMSD values ranging from 1.20-1.40, indicating relative similarities.
The left-handed superpositions matched with more trypsins and displayed better RMSD values ranging from 0.93-1.11.
The 3DS8 protein did not have many very specific active site matches with known proteins on the Chimera software [2], however, it was very similar to trypsin,
alpha-chymotrypsin, and proteinase B. The function of 3DS8 is most likely very similar to those since the active sites have the same amino acids and structures that differ within <4 angstroms.
The Dali database [3] was used to determine the conserved sequences within the 3ds8 protein. The majority of the hits were lipases with Z-scores up to 31.8, meaning the proteins are homologous
to the 3ds8 structure because they are higher than 20. Some hits had high LALI numbers that indicate matching residues in the structure. The active site residues are G104, S102, H222, D188. Each of
these is conserved in the 4 selected protein matches, which means that the active site is conserved. The 3ds8 active site is conserved in many other proteins with similar functions, many of which
are lipases. Since the 3DS8 has so many structural similarities to lipases, it most likely has the same functionality as the known proteins.
The BLAST database [4] was utilized to search for similar gene sequences and corresponding residue patterns. Using the FASTA sequence, the 3DS8 sequence is matched with similar proteins in an
alignment that shows similar positions of matching residues. The superfamily of 3ds8 is an αβ-hydrolase. Function and cellular position are unknown, but it is hydrolase-like and exists in bacteria.
The 3DS8 protein is part of the superfamily of alpha-beta hydrolases, so it most likely has the same function. Many structural and sequential similarities are conserved between 3DS8
and matched proteins, indicating that 3DS8 could very well be a hydrolase.
FASTA sequence of 3DS8:
KDQIPIILIHGSGGNASSLDKMADQLMNEYRSSNEALTMTVNSEGKIKFEGKLTKDAKRPIIKFGFEQNQATPDDWSKWLKIAMEDLKSRYGFTQMDGVGHSNGGLALTYYAEDYAGDKTVPTLRKLVAIGSPFNDLDPNDNGMDLSFKKLPNSTPQMDYFIKNQTEVSPDLEVLAIAGELSEDNPTDGIVPTISSLATRLFMPGSAKAYIEDIQVGEDAVHQTLHETPKSIEKTYWFLEKFKTDETVIQLDYK
InterPro Scan [5] searched for structure and taxonomy relations. The results have information about the domains and families of the protein. At the bottom, there are biological processes,
molecular functions, and cellular components to learn more about the protein and where it originates from. InterPro confirmed that 3DS8 is most likely a hydrolase. Since 3DS8 is part of
the hydrolase superfamily, its structure and function are likely to be that of some sort of hydrolase.
The last molecular modeling strategy was using SwissDock to investigate different ligands for 3DS8. Using the previously mentioned 4 residues that make up the active site, there were a few
ligands that demonstrated promising binding to 3DS8. The best choices would be PNP alpha-D-glucopyranoside or PNP N-acetyl-Beta-D-glucosaminide because they are close to the residues
of the active site.
The molecular weight of 3DS8 is proposed to be about 28 kDa.
