Optimized measurement temperature gives access to the solution structure of a 49 kDa homohexameric β-propeller.
Ph1500 is a homohexameric, two-domain protein of unknown function from the hyperthermophilic archaeon Pyrococcus horikoshii. The C-terminal hexamerization domain (Ph1500C) is of particular interest, as it lacks sequence homology to proteins of known structure. However, it resisted crystallization for X-ray analysis, and proteins of this size (49 kDa) present a considerable challenge to NMR structure determination in solution. We solved the high-resolution structure of Ph1500C, exploiting the hyperthermophilic nature of the protein to minimize unfavorable relaxation properties by high-temperature measurement. Thus, the side chain assignment (97%) and structure determination became possible at full proton density. To our knowledge, Ph1500C is the largest protein for which this has been achieved. To minimize detrimental fast water exchange of amide protons at increased temperature, we employed a strategy where the temperature was optimized separately for backbone and side chain experiments.
Varnay
Ilka
I
Institute for Advanced Study and Center of Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.
Truffault
Vincent
V
Djuranovic
Sergej
S
Ursinus
Astrid
A
Coles
Murray
M
Kessler
Horst
H
D000577:Amides / Q000737:chemistry; D018360:Crystallography, X-Ray; D009682:Magnetic Resonance Spectroscopy; D017433:Protein Structure, Secondary; D043902:Pyrococcus horikoshii / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D013696:Temperature
D000577:Amides; D012996:Solutions