title: Probing the solution structure of the DNA-binding protein Max by a combination of proteolysis and mass spectrometry.
abstract: A simple biochemical method that combines enzymatic proteolysis and matrix-assisted laser desorption ionization mass spectrometry was developed to probe the solution structure of DNA-binding proteins. The method is based on inferring structural information from determinations of protection against enzymatic proteolysis, as governed by solvent accessibility and protein flexibility. This approach was applied to the study of the transcription factor Max--a member of the basic/helix-loop-helix/zipper family of DNA-binding proteins. In the absence of DNA and at low ionic strengths, Max is rapidly digested by each of six endoproteases selected for the study, results consistent with an open and flexible structure of the protein. At physiological salt levels, the rates of digestion are moderately slowed; this and the patterns of cleavage are consistent with homodimerization of the protein through a predominantly hydrophobic interface. In the presence of Max-specific DNA, the protein becomes dramatically protected against proteolysis, exhibiting up to a 100-fold reduction in cleavage rates. Over a 2-day period, both complete and partial proteolysis of the Max-DNA complex is observed. The partial proteolytic fragmentation patterns reflect a very high degree of protection in the N-terminal and helix-loop-helix regions of the protein, correlating with those expected of a stable dimer bound to DNA at its basic N-terminals. Less protection is seen at the C-terminal where a slow, sequential proteolytic cleavage occurs, correlating to the presence of a leucine zipper. The results also indicate a high affinity of Max for its target DNA that remains high even when the leucine zipper is proteolytically removed. In addition to the study of the helix-loop-helix protein Max, the present method appears well suited for a range of other structural biological applications.
authors.affiliation: Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York 10021, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Cohen
authors.forename: A R
authors.initials: AR
authors.lastname: Ferré-D'Amaré
authors.forename: S K
authors.initials: SK
authors.lastname: Burley
authors.forename: B T
authors.initials: BT
authors.lastname: Chait
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D016350:Leucine Zippers; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010449:Peptide Mapping; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000378:metabolism; D012996:Solutions; D013381:Subtilisins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C067850:Myc associated factor X; D012996:Solutions; D014157:Transcription Factors; D012697:Serine Endopeptidases; D013381:Subtilisins; C028344:glutamyl endopeptidase