Alanine mutagenesis of high-mobility-group-protein-1 box B (HMG1-B).
We have generated a set of alanine-scanning substitutions in high-mobility-group protein 1 box B (HMG1-B; the second domain of the HMG1 nuclear protein from the rat) in order to explore the influence of specific surface side chains on its function and folding. Guanidine hydrochloride and thermal unfolding studies have been carried out to investigate the effect of substituted residues on the folding pathway. Binding to four-way junction and linear-duplex DNA has been assayed to determine which residues play an important role in DNA binding. We have identified several mutants that are more stable or bind more tightly to the junction than the wild-type, including the particular phenylalanine side chain that is thought to intercalate into the DNA. Thus the interaction between HMG1-B and branched DNA substrates should exhibit differences from present models based on the structure of the complexes that have been solved to date.
Taudte
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S
Department of Chemistry, New York University, 31 Washington Place, New York, NY 10003, USA.
Xin
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H
Kallenbach
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D000409:Alanine / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D051381:Rats; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D010649:Phenylalanine; D014364:Tryptophan; D004247:DNA; D019791:Guanidine; D000409:Alanine