W323S variant of Xiap-Bir3 binds to SMAC but not caspase-9

The ability of the wild-type XIAP BIR3 domain as well as its Trp323Ser variant in inhibition of human caspase-9, binding to AVPFVASLPN (SMAC-peptide), SMAC protein, and mature caspase-9 was investigated. In order to investigate the role of W323 on these interactions, this residue was mutated to Serine. Circular dichroism as well as thermal denaturation studies showed that W323S mutation did not hamper proper folding of the protein. The dissociation constants for the interaction of the wild type BIR3 as well as its mutant to Smac-type peptide were found to be 1.8 and 27 muM, respectively. The inhibition of and binding to caspase-9 by wild-type BIR3 and its mutant were also compared. While the wild-type protein potently inhibited the enzyme, the mutant failed to do so. The lack of caspase-9 inhibition was due to absence of interaction of the mutant BIR3 with mature caspase-9. These results indicate that Trp323 of BIR3 plays a pivotal role both in maintaining necessary conformation for caspase-9 interaction and to a lesser extent, recognition of Smac-type peptide. Moreover, decreased stability of the mutant compared with the wild type indicates that W323 is essential for maintaining the stability BIR3-Smac-peptide complex.     

Structural studies of a two-domain chitinase from Streptomyces griseus HUT6037

Chitinase C (ChiC) from Streptomyces griseus HUT6037 was the first glycoside hydrolase family 19 chitinase that was found in an organism other than higher plants. An N-terminal chitin-binding domain and a C-terminal catalytic domain connected by a linker peptide constitute ChiC. We determined the crystal structure of full-length ChiC, which is the only representative of the two-domain chitinases in the family. The catalytic domain has an alpha-helix-rich fold with a deep cleft containing a catalytic site, and lacks three loops on the domain surface compared with the catalytic domain of plant chitinases. The chitin-binding domain is an all-beta protein with two tryptophan residues (Trp59 and Trp60) aligned on the surface. We suggest the binding mechanism of tri-N-acetylchitotriose onto the chitin-binding domain on the basis of molecular dynamics (MD) simulations. In this mechanism, the ligand molecule binds well on the surface-exposed binding site through two stacking interactions and two hydrogen bonds and only Trp59 and Trp60 are involved in the binding. Furthermore, the flexibility of the Trp60 side-chain, which may be involved in adjusting the binding surface to fit the surface of crystalline chitin by the rotation of chi2 angle, is shown.     

Mutational analysis of a CBM family 5 chitin-binding domain of an alkaline chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.     

Tryptophan in alpha-helix 3 of Tet repressor forms a sequence-specific contact with tet operator in solution

The Tn10-encoded Tet repressor contains two tryptophan residues at positions 43 and 75. The typical tryptophan fluorescence is decreased upon binding of tet operator. The Tet repressor gene was engineered to replace either or both of the Trp codons by Phe codons. The resulting single tryptophan mutants are called F43 and F75 and the double mutant F43F75. The mutant proteins were purified to homogeneity. They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. F75 and wild-type bind tet operator with the same association constant. The association constants of F43 and F43F75 with tet operator are about 3 orders of magnitude smaller. This indicates that Trp43 is important for tet operator recognition. Trp43 fluorescence is completely quenched in the complex with tet operator DNA while Trp75 remains unaffected. Binding to nonspecific DNA leads only to a 40% decrease of Trp43 fluorescence. This is interpreted as the contribution of the changed environment while the complete quench reflects a tight sequence-specific contact of tryptophan 43 to tet operator DNA. Trp43 is solvent-exposed, while Trp75 is buried in the hydrophobic interior of the protein. These results are discussed in light of the alpha-helix turn-alpha-helix DNA binding motif deduced from homology to other repressor proteins.     

Orientation of alpha-neurotoxin at the subunit interfaces of the nicotinic acetylcholine receptor

The alpha-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. Because the alpha-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.     

High‐energy water sites determine peptide binding affinity and specificity of PDZ domains

PDZ domains have well known binding preferences for distinct C‐terminal peptide motifs. For most PDZ domains, these motifs are of the form [S/T]‐W‐[I/L/V]. Although the preference for S/T has been explained by a specific hydrogen bond interaction with a histidine in the PDZ domain and the (I/L/V) is buried in a hydrophobic pocket, the mechanism for Trp specificity at the second to last position has thus far remained unknown. Here, we apply a method to compute the free energies of explicit water molecules and predict that potency gained by Trp binding is due to a favorable release of high‐energy water molecules into bulk. The affinities of a series of peptides for both wild‐type and mutant forms of the PDZ domain of Erbin correlate very well with the computed free energy of binding of displaced waters, suggesting a direct relationship between water displacement and peptide affinity. Finally, we show a correlation between the magnitude of the displaced water free energy and the degree of Trp‐sensitivity among subtypes of the HTRA PDZ family, indicating a water‐mediated mechanism for specificity of peptide binding.     

The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.     

Mutational Analysis of a CBM Family 5 Chitin-Binding Domain of an Alkaline Chitinase from Bacillus sp. J813

Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions.     

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70-86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.     

Detection and characterization of two ATP-dependent conformational changes in proteolytically inactive Escherichia coli Lon mutants by stopped flow kinetic techniques

Lon is an ATP dependent serine protease responsible for degrading denatured, oxidatively damaged and certain regulatory proteins in the cell. In this study we exploited the fluorescence properties of a dansylated peptide substrate (S4) and the intrinsic Trp residues in Lon to monitor peptide interacting with the enzyme. We generated two proteolytically inactive Lon mutants, S679A and S679W, where the active site serine is mutated to an Ala and Trp residue, respectively. Stopped-flow fluorescence spectroscopy was used to identify key enzyme intermediates generated along the reaction pathway prior to peptide hydrolysis. A two-step peptide binding event is detected in both mutants, where a conformational change occurs after a rapid equilibrium peptide binding step. The Kd for the initial peptide binding step determined by kinetic and equilibrium binding techniques is approximately 164 micromolar and 38 micromolar, respectively. The rate constants for the conformational change detected in the S679A and S679W Lon mutants are 0.74 +/- 0.10 s(-1) and 0.57 +/- 0.10 s(-1), respectively. These values are comparable to the lag rate constant determined for peptide hydrolysis (klag approximately 1 s(-1)) [Vineyard, D., et al. (2005) Biochemistry 45, 4602-4610]. Replacement of the active site Ser with Trp (S679W) allows for the detection of an ATP-dependent conformational change within the proteolytic site. The rate constant for this conformational change is 7.6 +/- 1.0 s(-1), and is essentially identical to the burst rate constant determined for ATP hydrolysis under comparable reaction conditions. Collectively, these kinetic data support a mechanism by which the binding of ATP to an allosteric site on Lon activates the proteolytic site. In this model, the energy derived from the binding of ATP minimally supports peptide cleavage by allowing peptide substrate access to the proteolytic site. However, the kinetics of peptide cleavage are enhanced by the hydrolysis of ATP.     

Insights into a conformational epitope of Hev b 6.02 (hevein)

Hevein (Hev b 6.02) is a major IgE-binding allergen in natural rubber latex and manufactured products. Both tryptophans (Trp(21) and Trp(23)) of the hevein molecule were chemically modified with BNPS-skatole (2-nitrophenylsulfenyl-3-methyl-3(')-bromoindolenine); derivatized allergen failed to significantly inhibit binding of serum IgE in ELISA assays. Similarly, skin prick tests showed that hevein-positive patients gave no response with the modified allergen. Dot blot experiments carried out with anti-hevein mono- and polyclonal antibodies confirmed the importance of Trp(21) and Trp(23) for antibody-recognition, and demonstrated the specific cross-reactivity of other molecules containing hevein-like domains. We also report the structure of Hev b 6.02 at an extended resolution (1.5A) and compare its surface properties around Trp residues with those of similar regions in other allergens. Overall our results indicate that the central part of the protein, which comprises three aromatic and other acidic and polar residues, constitutes a conformational epitope.     

The C terminus of sigma(32) is not essential for degradation by FtsH

A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.     

Analysis of the substrate binding sites of human galactosyltransferase by protein engineering.

An expression vector, pIN-GT, encoding the soluble form of beta 1,4-galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN-III-ompA2 expression vector. Escherichia coli strain SB221 harboring the pIN-GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT. The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti-GT antibodies. The recombinant GT was purified to homogeneity by N-acetylglucosamine-Sepharose affinity chromatography. The NH2-terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase. This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites. Photoaffinity labeling of GT with UDP-galactose analog, 4-azido-2-nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328. Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site-directed mutagenesis. Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287----Phe remained as active as wild-type GT. Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N-acetyglucosamine binding and Tyr309 is involved in UDP-galactose binding as well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

The Cel5 cellulase (formerly known as endoglucanase Z) from Erwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBD(Cel5) has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBD(Cel5) exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBD(Cel5) bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.     

Kinetic coupling of folding and prolyl isomerization of beta2-microglobulin studied by mutational analysis

β₂-Microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of β2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of β2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V β2-ms) as well as the wild-type β2-m. W39 β2-m is a triple mutant in which both of the authentic Trp residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 β2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V β2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic coupling of unfolding/refolding and prolyl isomerization, which has tended to be neglected in recent studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance.     

Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides

Arthropod hemocyanins and phenoloxidases serve different physiological functions as oxygen transporters and enzymes involved in defense reactions, respectively. However, they are equipped with a structurally similar oxygen-binding center. We have shown that the clotting enzyme of the horseshoe crab, Tachypleus tridentatus, functionally converts hemocyanin to phenoloxidase by forming a complex without proteolytic cleavage (Nagai, T., and Kawabata, S. (2000) J. Biol. Chem. 275, 35297-35301). Here we show that chitin-binding antimicrobial peptides of the horseshoe crab induce the intrinsic phenoloxidase activity of hemocyanin. Tachyplesin, a major Tachypleus antimicrobial peptide with an amphiphilic structure, converted the hemocyanin to phenoloxidase. Surface plasmon resonance analysis revealed the specific interaction of tachyplesin with hemocyanin at K(d) = 3.4 x 10(-)6 m. The chemical modification of Trp or Tyr in tachyplesin, but not Lys or Arg, dramatically reduced the affinity to hemocyanin, suggesting that the binding site is located in the hydrophobic face of tachyplesin. Hemocyanin has no affinity with chitin, but it significantly binds to tachyplesin-coated chitin, leading to the expression of phenoloxidase activity. The chitin coated with antimicrobial peptides may serve as a scaffold for the binding of hemocyanin, and the resulting phenoloxidase activity appears to function as a trigger of exoskeleton wound healing.     

Lipid-protein interactions: NMR study of melittin and its binding to lysophosphatidylcholine.

Proton NMR of melittin differs according to the association state of the peptide in the monomer or tetramer. Melittin interacts with lysophosphatidyl-choline micelles, whatever the association state of melittin; well resolved superimposed spectra from both components for all the lipid to peptide molar ratios are observed. Within the complexes, local mobility and fast exchange occurs. On binding concomitant shifts on Trp19 indole lines and on the aliphatic CH2 protons of the lipids are detected. The lipid perturbation is maximum for methylene groups in a alpha and beta of the ester bond, this could allow positionning of Trp19 in the hydrophobic core of the lipids.     

Interaction of monoclonal anti-peptide antibodies with lysozyme

The interaction of monoclonal anti-peptide antibodies with the free peptide and its protein counterpart has been evaluated for hen egg white lysozyme and the peptide constituting residues 38 to 45. Fluorescence methodology has been developed for the measurement of association constants based on resonance energy transfer between the excited tryptophan of antibody and bound peptide ligand conjugated to a fluorescent probe. Five antibodies, four IgM and one IgG, have been assayed by ELISA, and have demonstrated binding to the adsorbed peptide alone, to the adsorbed lysozyme alone, or to both. Multivalent interaction with the adsorbed ligand is a key factor in the efficacy of binding. Measurement of binding constants in homogeneous solution, by equilibrium dialysis and energy transfer, demonstrated that lysozyme was bound to an IgG antipeptide antibody with an association constant (4 X 10(2) M-1) 200-fold less than that for the free peptide (8 X 10(4) M-1). It was also inferred for IgM that an association constant of the order of 10(2) M-1 was sufficient to effect selective interaction in a system providing multivalent interaction. The shared conformations between protein and peptide, implied by the specific reactivity of the anti-peptide antibody with the protein, points to structural fluctuations of the surface regions and residues of globular proteins.     

Biophysical characterization of the interaction of p21 with calmodulin: a mechanistic study

p21 is a protein with important roles in cell proliferation, cell cycle regulation and apoptosis. Several studies have demonstrated that its intracellular localization plays an important role in the functional regulation and binding of calmodulin favors its nuclear translocation. However, the detail mechanism of the interaction with p21 and calmodulin is not well understood. In this report, peptides derived from the C-terminal of p21 that cover the binding domain of calmodulin were used to investigate the association of p21 with calmodulin. We found p21(141-164) interaction with Ca(2+)-saturated dansyl-labelled calmodulin caused a significant increase in dansyl fluorescence intensity and a blue shift of the maximum emission from 510 to 475 nm. The Trp fluorescence intensities of mutated p21(141-164) peptides (F150W, Y151W and F159W) increased upon binding to Ca(2+)-saturated calmodulin and fluorescence maxima were blue shifted from 350 nm to 330 nm. The results suggested p21(141-164) is most likely buried in the hydrophobic binding tunnel of calmodulin. Both dansyl and Trp fluorescence titrations generated dissociation constants around 0.1 muM and a stoichiometry of 1:1, which was further confirmed by nondenaturing gel band shift electrophoresis. Fluorescence titrations and Trp fluorescence quenching results indicated electrostatic interaction is involved in this association. Upon binding to calmodulin, p21(141-164) remained largely unstructured and showed only about 15% alpha-helix. In contrast to other calmodulin binding peptide, the dominant force in the association of p21(141-164) with calmodulin may be electrostatic interaction. Our results would be helpful for understanding the molecular details of p21 and calmodulin interaction.     

Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.