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At least 19 recordsLinked to original sources
PubMed2003

Structural analysis of gelsolin using synchrotron protein footprinting.

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.

Calcium↗
PubMed2005

Radiolytic modification of sulfur-containing amino acid residues in model peptides: fundamental studies for protein footprinting.

Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using gamma-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the gamma-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine approximately tryptophan > cystine is observed.

Amino Acid Sequence↗
PubMed1994

Protein footprinting by the combined use of reversible and irreversible lysine modifications.

A two-step lysine-modification procedure has been devised to chemically footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a complex, is first treated lightly with a reversible lysine-modifying reagent. The protein is then unfolded and treated extensively with an irreversible lysine reagent to block those lysines that did not react previously; next, the first lysine modification is reversed, and a lysine-specific endoproteinase is used to cleave the tagged polypeptide at the deblocked lysines. Separation of the proteolytic products by size and identification of the tagged fragments map the positions of these lysines. In this procedure, the reversible lysine reagent serves as the chemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macromolecular contacts are identified from differences in proteolytic patterns of the tagged protein when the first lysine modification is done with the protein in the free form and in a complex. Application of the method to vaccinia virus topoisomerase identifies a number of lysines that are involved in its binding to DNA.

Amino Acid Sequence↗
PubMed1998

Fluorescence-based protein footprinting using histidine-tagged protein.

We describe a procedure for protein footprinting to identify the region(s) of a protein that interacts with a ligand. The method utilized the affinity of a stretch of histidine residues cloned into the protein to metal-chelated resin. After limited protease digestion, the histidine-tagged end fragments were separated by the resin and labeled with a fluorescein derivative. Resolving the labeled digestion products on a denaturing polyacrylamide gel and visualizing the peptides using a FluorImager provided a way to identify the protease target sites that were protected from digestion because of interaction with DNA. The protection experiments would be applicable not only to detect direct contact sites but also sites allosterically altered by ligand binding.

Chromatography, Affinity↗
PubMed1988

Protein antigen-monoclonal antibody contact sites investigated by limited proteolysis of monoclonal antibody-bound antigen: protein "footprinting".

This study describes the use of limited proteolysis of monoclonal antibody (mAb)-bound antigens in the analysis of the two measles virus surface glycoproteins. This approach is dubbed protein "footprinting" in analogy with DNA "footprinting." Protein footprinting was superior to competitive-binding assays and as good as in vitro mAb-selected variant analysis in differentiating among mAbs with various specificities to a given protein. Proteolytic digestion of the antigen prior to mAb binding drastically reduced mAb binding resulting in poor differentiation among mAbs. In contrast, protein footprinting showed that some mAbs retained the ability to immunoprecipitate such fragments. Thus footprinting could be used for localization of mAb epitopes on a protein and proved also to be an effective means of distinguishing among mAb-selected variants differing in single epitopes. Conformational changes caused by heat-denaturation or the binding of anti-antibody to an antigen-antibody complex could also be detected by footprinting.

Antibodies, Monoclonal↗
PubMed2004

Mapping protein-ligand interactions by hydroxyl-radical protein footprinting.

Hydroxyl-radical protein footprinting is a direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end-label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high-resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that show regions of the protein that change their susceptibility to cleavage while bound to a ligand.

Amino Acid Sequence↗
PubMed1994

Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach.

A novel direct approach, analogous to DNA footprinting, for mapping protein domains involved in macromolecular interactions is presented in this paper and applied to cAMP receptor protein (CRP) interactions with the allosteric ligand (cAMP) and DNA. In this approach, a protein-macromolecule complex is subjected to a nonspecific cleavage by Fe-EDTA. The cleavage products are resolved by SDS-PAGE and transferred to a PVDF membrane. Transferred polypeptides are visualized by immunostaining with antibodies specific to the N-terminal peptide of the protein. The mobility of the bands visualized in such a way is directly proportional to the distance of the cleavage sites from the N-terminus, and thus the positions of the sites protected from cleavage by a bound macromolecule can be determined. Thus, protein domains involved in macromolecular interactions can be mapped. In the case of CRP, the cleavage conditions were established which resulted in, on the average, less than one cleavage event/protein molecule and which preserved satisfactory levels of protein and DNA activity. When applied to CRP-DNA interactions, the protein footprinting approach correctly identified domains of CRP that were known to be involved in the recognition of DNA. The obtained results showed also that the binding of CRP to the DNA binding site perturbed the region of CRP involved in intersubunit interactions. An allosteric ligand (cAMP) appeared to perturb the same region of CRP. This stresses out the importance of intersubunit interactions in cAMP modulation of protein DNA binding affinity. The protein footprinting methodology presented in this paper should be broadly generalizable to any protein-macromolecule system.

Amino Acid Sequence↗
PubMed1997

Mapping conformational changes in a protein: application of a protein footprinting technique to cAMP-induced conformational changes in cAMP receptor protein.

We have used protein footprinting [Heyduk, E., & Heyduk, T. (1994) Biochemistry 33, 9643] to detect and map ligand-induced conformational changes in cAMP receptor protein (CRP). The binding of cAMP to CRP dramatically increases the specific DNA binding activity of the protein and, as has been previously shown, induces conformational changes in the protein. Protein footprinting experiments with the free CRP, the CRP-cAMP complex, and the CRP-cGMP complex were analyzed quantitatively. Binding of cAMP produced measurable differences in the susceptibility of CRP to the cleavage by Fe-EDTA. Almost all of these changes occurred in the C-terminal domain (DNA binding domain) of the protein. Additional changes were observed at the ends of the C alpha-helix, which is involved in intersubunit contacts in the CRP dimer, and in the hinge peptide, connecting N-terminal and C-terminal domains of the protein. The boundaries of the regions in the C-terminal domain, which exhibited changes in susceptibility to Fe-EDTA cleavage, almost exactly corresponded to D, E, and F alpha-helices which are involved directly in the recognition of DNA. The F alpha-helix, which provides all base-specific contacts in the CRP-DNA complex, became hypersensitive to Fe-EDTA-mediated cleavage, whereas the solvent exposure of D and E alpha-helices was decreased upon binding of cAMP. These results suggest that a significant part of cAMP-induced conformational change in CRP involves a movement of secondary structure elements in the C-terminal domain of the protein so that the recognition F alpha-helix becomes exposed to the solvent. In contrast to cAMP, binding of cGMP produced insignificant changes in susceptibility to Fe-EDTA-mediated cleavage. This is consistent with the inability of cGMP to induce functional conformational changes in CRP. The protein footprinting technique appears to be sufficiently sensitive for detection and mapping of ligand-induced conformational changes in proteins.

Binding Sites↗
PubMed1999

Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting.

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.

Amino Acid Sequence↗
PubMed2001

Mapping DNA-binding sites of HIV-1 integrase by protein footprinting.

The HIV-1 integrase protein catalyzes integration of the viral genome into host cell DNA. Whereas the structures of the three domains of integrase have been solved separately, both the structural organization of the full-length protein and its interaction with DNA remain unresolved. A protein footprinting approach was employed to investigate the accessibility of residues in the full-length soluble integrase mutant, INF(185K,C280S), to proteolytic attack in the absence and presence of DNA. The N-terminal and C-terminal domains were relatively more accessible to proteolytic attack than the core domain. The susceptibility to proteolytic attack was specifically affected by DNA at residues Lys34, in the N-terminal domain, Lys111, Lys136, Glu138, Lys156-Lys160, Lys185-Lys188, in the core domain, and Asp207, Lys 215, Glu246, Lys258 and Lys273 in the linker and C-terminal domain, suggesting that these regions are involved in, or shielded by, DNA binding. Lys34 is positioned in a putative dimerization domain, consistent with the notion that DNA stabilizes the dimeric state of integrase.

Amino Acid Sequence↗
PubMed2005

Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting.

Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectable products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by gamma-rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectable. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise approximately 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.

Amino Acids↗
PubMed1999

Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases.

CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein. We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830-10836]. In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe-DTPA]2-), iron complexed to ethylenediaminediacetic acid ([Fe-EDDA]), iron complexed to desferrioxamine mesylate ([Fe-HDFO]+), and copper complexed to o-phenanthroline ([(OP)2Cu]+) as proteases. These chemical proteases differ in size, charge, and hydrophobicity. Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe-EDDA] and [Fe-DTPA]2- of CRP-cAMP detected hypersensitivities in the DNA-binding F alpha-helix, the interdomain hinge, and the ends of the C alpha-helix, which is involved in intersubunit interactions. [Fe-EDDA] and [Fe-DTPA]2- also detected reductions in cleavage in the D and E alpha-helices, which are involved in DNA recognition. Cleavage by [Fe-HDFO]+ of CRP-cAMP detected hypersensitivities in beta-strand 8, the B alpha-helix, as well as in parts of the F and C alpha-helices. [Fe-HDFO]+ also detected protections from cleavage in beta-strands 4 to 5 and their intervening loop, beta-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E alpha-helices. Cleavage by [(OP)2Cu]+ of CRP-cAMP detected hypersensitivities in beta-strands 9 and 11 as well as in the D and E alpha-helices. [(OP)2Cu]+ also detected protections in the C alpha-helix , the interdomain hinge, and beta-strands 2-7. Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by [(OP)2Cu]+, which detected minor protections in beta-strands 3-7, the interdomain hinge, and the C alpha-helix. These results show that binding of cAMP causes structural changes in CRP in the nucleotide binding domain, the interdomain hinge, the DNA binding domain, and regions involved in intersubunit interaction. Structural changes induced by binding of cGMP appear to be very minor and confined to the nucleotide binding domain, the interdomain hinge, and regions involved in intersubunit interaction. Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single protease alone.

Cyclic AMP↗
PubMed2004

Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting.

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.

Animals↗
PubMed2002

Correlating protein footprinting with mutational analysis in the bacterial transcription factor sigma54 (sigmaN).

Protein footprints of the enhancer-dependent sigma54 protein, upon binding the Escherichia coli RNA polymerase core enzyme or upon forming closed promoter complexes, identified surface-exposed residues in sigma54 of potential functional importance at the interface between sigma54 and core RNA polymerases (RNAP) or DNA. We have now characterised alanine and glycine substitution mutants at several of these positions. Properties of the mutant sigma54s correlate protein footprints to activity. Some mutants show elevated DNA binding suggesting that promoter binding by holoenzyme may be limited to enable normal functioning. One such mutant (F318A) within the DNA binding domain of sigma54 shows a changed interaction with the promoter regulatory region implicated in transcription silencing and fails to silence transcription in vitro. It appears specifically defective in preferentially binding to a repressive DNA structure believed to restrict RNA polymerase isomerisation and is largely intact for activator responsiveness. Two mutants, one in the regulatory region I and the other within core interacting sequences of sigma54, failed to stably bind the activator in the presence of ADP-aluminium fluoride, an analogue of ATP in the transition state for hydrolysis. Overall, the data presented describe a collection sigma54 mutants that have escaped previous analysis and display an array of properties which allows the role of surface-exposed residues in the regulation of open complex formation and promoter DNA binding to be better understood. Their properties support the view that the interface between sigma54 and core RNAP is functionally specialised.

Amino Acid Substitution↗
PubMed1996

Sequence-specific DNA binding of the phage Mu C protein: footprinting analysis reveals altered DNA conformation upon protein binding.

The mom gene of bacteriophage Mu, which codes for a DNA modification function, is regulated in a complex manner at both transcriptional and translational levels. The phage-encoded C protein functions as an activator of mom transcription. The mom promoter has features of an activator-dependent weak promoter, and the C binding site is located upstream and overlapping the -35 region and includes the palindromic sequence TTAT(N)6ATAA. The interactions of this activator protein at its binding site in Pmom has been investigated using four different chemical footprinting reagents. The protein footprint spans a region of 18 to 25 bp, depending on the nature of the chemical reagent used. Dimethylsulfate protection experiments revealed the base-specific interactions. The protected guanines are separated by 15 bp and are located beyond the interrupted palindromic sequence. A tripartite footprint was observed with hydroxyl radical, generated by Fe(II)-EDTA, which shows the binding of the protein to one face of the helix. The extent of protection conferred by the bound protein, however, is not uniform, suggesting that the interaction is asymmetric. The chemical nuclease 1,10-phenanthroline-copper, a minor groove specific ligand, shows hyper-reactivity upon protein binding in the top strand nucleotide triplet CAC, again confirming the protein-induced alterations in DNA conformation. Gel exclusion chromatography and chemical crosslinking experiment with the purified protein suggest that this mode of interaction is accomplished by a dimeric protein. This observation is supported by electrophoretic mobility shift assay using heterodimer of pure C protein and staphylococcal protein A-C fusion. The deletion analysis implicates a role for the carboxyl-terminal region of the protein in DNA binding.

Base Sequence↗
PubMed1995

Intermolecular binding sites of human immunodeficiency virus type 1 Rev protein determined by protein footprinting.

Human immunodeficiency virus encodes the regulatory protein Rev, which is required for expression of viral structural proteins. It binds to an RNA element (RRE) in the viral transcript and up-regulates the cytoplasmic appearance of unspliced and singly spliced viral mRNA. We have studied the structure of Rev alone and complexed with the RRE and two monoclonal antibodies, using a protein footprinting approach. The method involves radioactive labeling at the C-terminal end of Rev fusion protein followed by limited proteolysis under native conditions, using 10 different proteinases. Rev protein was mainly cleaved within the basic domain and in the C-terminal part. The periodicity of the proteolytic cleavages within the basic domain strongly suggests that it forms an alpha-helical structure with one side facing the solvent. In the presence of RRE, these cleavages became significantly reduced. In addition, strong protection was observed at position 66 outside the basic domain. As a control for the specificity of the footprinting reaction, we confirmed the position of the epitopes for two monoclonal antibodies. This protein footprinting methodology is generally applicable to other proteins for which terminal modifications are acceptable, and provides a useful tool for mapping structure, substrate binding, and conformational changes.

Base Sequence↗
PubMed2002

Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting.

Cofilin is an actin regulatory protein that binds to both monomeric and filamentous actin, and has filament severing activity. Although crystal structures for the monomeric forms of both G-actin and cofilin have been described, the structure of the binary cofilin-G-actin complex is not available. Synchrotron protein footprinting is used to identify specific side chain residues on the cofilin surface that are buried in the formation of the cofilin-G-actin binary complex. Exposure to synchrotron X-rays results in stable oxidative modifications of aromatic, aliphatic, and sulfur-containing side chains, with the rate of modification for a particular residue being dependent on its intrinsic reactivity and solvent accessibility. The rates of modification were monitored for a number of peptides generated by digestion of oxidized cofilin, both in isolation and in its binary complex with G-actin. After binding to G-actin takes place, a significant decrease in modification rates, indicating protection of side chain groups, is seen for cofilin peptides corresponding to residues 4-20, 10-17, 83-96, 91-105, and 106-117. A number of other peptides show no change in reactivity, and are presumed to represent regions distal to the binding site. Tandem mass spectrometry demonstrates that residues Leu 13, Pro 94, Met 99, and Leu 108 and 112 directly participate in the binding interface. These results are generally consistent with, and complementary to, the results of previous site-directed mutagenesis studies and extend our understanding of the G-actin binding surface of cofilin.

Actin Depolymerizing Factors↗
PubMed1998

Protein footprinting reveals specific binding modes of a high mobility group protein I to DNAs of different conformation.

The high mobility group proteins I and Y (HMGI/Y) are abundant components of chromatin. They are thought to derepress chromatin, affect the assembly and activity of the transcriptional machinery, and associate with constitutive heterochromatin during mitosis. HMGI/Y protein molecules contain three potential DNA-binding motifs (AT-hooks), but the extent of contacts between DNA and the entire protein has not been determined. We have used a protein-footprinting procedure to map regions of the Chironomus HMGI protein molecule that are involved in contacts with DNA. We find that in the presence of double-stranded DNA all AT-hook motifs are protected against hydroxyl radical proteolysis. In contrast, only two motifs were protected in the presence of four-way junction DNA. Large regions that flank the AT-hook motifs were found to be strongly protected against proteolysis in complexes with interferon-beta promoter DNA, suggesting amino acid residues outside the AT-hooks considerably contribute to DNA binding.

Animals↗