A characterization of the lumenal region of human tapasin reveals the presence of two structural domains.
Tapasin is a type I membrane glycoprotein involved with other accessory proteins in the assembly of class I MHC-beta(2)m-peptide complexes in the endoplasmic reticulum. We have probed the three-dimensional structure of the lumenal region of human tapasin (residues 1-392) tagged with a (His)(6) sequence at its C-terminus using biochemical and biophysical techniques. The far-UV circular dichroism spectrum revealed that tapasin possesses well-defined secondary structural elements corresponding predominantly to beta-sheets. A thermal denaturation curve recorded at 216 nm showed a midpoint transition centered at approximately 45 degrees C. Sedimentation analysis showed that tapasin is monomeric in solution with a sedimentation coefficient, S degrees (20,w), of 2.68 S. This value of S degrees (20,w) combined with the value of the molar mass obtained by MALDI mass spectrometry (44.2 kDa) yielded a frictional ratio, f/f(0), of 1.47. Assuming tapasin is a prolate ellipsoid, we calculated an apparent length of 22.5 nm and a diameter of 2.62 nm, consistent with an elongated molecular shape. Controlled proteolysis using various enzymes revealed that a narrow region of tapasin near residue 90 is highly susceptible to digestion, resulting in two fragments that are resistant to further cleavage. The identity of these fragments was determined by amino acid sequencing and MALDI mass spectrometry and revealed a 9 kDa N-terminal fragment and a 34 kDa C-terminal fragment. Collectively, these results suggest that tapasin is comprised of two core domains of different sizes loosely linked by a flexible region.
Chen
Mingnan
M
School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269, USA.
Stafford
Walter F
WF
Diedrich
Gundo
G
Khan
Amir
A
Bouvier
Marlene
M
D017920:Antiporters / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002918:Chymotrypsin / Q000737:chemistry; D002942:Circular Dichroism; D006801:Humans; D006868:Hydrolysis; D007136:Immunoglobulins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D026901:Membrane Transport Proteins; D010196:Pancreatic Elastase / Q000737:chemistry; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D012995:Solubility; D013820:Thermolysin / Q000737:chemistry; D014461:Ultracentrifugation
D017920:Antiporters; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C107762:tapasin; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D013820:Thermolysin