The HU protein from Thermotoga maritima: recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein.
The histone-like protein TmHU from the hyperthermophilic eubacterium Thermotoga maritima was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and cation exchange chromatography. CD spectroscopical studies with secondary structure analysis as well as comparative modeling demonstrate that the dimeric TmHU has a tertiary structure similar to other homologous HU proteins. The Tm of the protein was determined to be 96 degrees C, and thermal unfolding is nearly completely reversible. Surface plasmon resonance measurements for TmHU show that the protein binds to DNA in a highly cooperative manner, with a KD of 73 nM and a Hill coefficient of 7.6 for a 56 bp DNA fragment. It is demonstrated that TmHU is capable to increase the melting point of a synthetic, double-stranded DNA (poly[d(A-T)]) by 47 degrees C, thus suggesting that DNA stabilization may be a major function of this protein in hyperthermophiles. The significant in vitro protection of double-helical DNA may be useful for biotechnological applications.
Esser
D
D
Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Saale, Germany. dirk.esser@biochemtech.uni-halle.de
Rudolph
R
R
Jaenicke
R
R
Böhm
G
G
D000495:Allosteric Site; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D007526:Isoelectric Point; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000737:chemistry*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C040330:histone-like protein HU, bacteria; D004247:DNA