Low Dissociation Rate Constants Research Articles

Enhanced mechanosensing of cells in synthetic 3D matrix with controlled biophysical dynamics

3D culture of cells in designer biomaterial matrices provides a biomimetic cellular microenvironment and can yield critical insights into cellular behaviours not available from conventional 2D cultures. Hydrogels with dynamic properties, achieved by incorporating either degradable structural components or reversible dynamic crosslinks, enable efficient cell adaptation of the matrix and support associated cellular functions. Herein we demonstrate that given similar equilibrium binding constants, hydrogels containing dynamic crosslinks with a large dissociation rate constant enable cell force-induced network reorganization, which results in rapid stellate spreading, assembly, mechanosensing, and differentiation of encapsulated stem cells when compared to similar hydrogels containing dynamic crosslinks with a low dissociation rate constant. Furthermore, the static and precise conjugation of cell adhesive ligands to the hydrogel subnetwork connected by such fast-dissociating crosslinks is also required for ultra-rapid stellate spreading (within 18 h post-encapsulation) and enhanced mechanosensing of stem cells in 3D. This work reveals the correlation between microscopic cell behaviours and the molecular level binding kinetics in hydrogel networks. Our findings provide valuable guidance to the design and evaluation of supramolecular biomaterials with cell-adaptable properties for studying cells in 3D cultures.

Interactions between a Bioflavonoid and c-MYC Promoter G-Quadruplex DNA: Ensemble and Single-Molecule Investigations.

Small molecules capable of stabilizing the G-quadruplex structure of the nuclease hypersensitivity element III1 (NHE III1) are useful in controlling the overexpression of the c-MYC oncogene. In this study, we have probed the interactions of a 22-mer c-MYC promoter quadruplex-forming sequence (Pu22) with a bioflavonoid 3,4',5,7-tetrahydroxyflavone, commonly known as kaempferol (KF). Ensemble fluorescence resonance energy transfer experiments on labeled Pu22 indicate that KF decreases the affinity of the former toward its complimentary strand, suggesting the stabilization of the quadruplex structure of Pu22. Considering that binding dynamics plays an important role in supramolecular interactions, there is hardly any information on this aspect for quadruplex-flavonoid systems; we have studied the kinetics of KF-Pu22 complexation and decomplexation processes on the single-molecule level by employing fluorescence correlation spectroscopy technique. The binding dynamics is characterized by a fast relaxation time of 10-50 μs. This leads to a high association rate constant ( k+) of ∼109 M-1 s-1, which is close to the pure diffusion controlled limit. However, it is the low dissociation rate constant ( k-) of ∼104 s-1 that is mainly responsible for the stability of the KF-Pu22 complex. Molecular docking study shows that KF binds near the 3'-end of Pu22 by forming several H-bonds with the bases. These findings suggest that KF is a potential binder of the c-MYC promoter quadruplex DNA and can be useful in anticancer therapies.

Noncatalytic Endosialidase Enables Surface Capture of Small-Cell Lung Cancer Cells Utilizing Strong Dendrimer-Mediated Enzyme-Glycoprotein Interactions.

Enumeration of circulating tumor cells (CTCs) of small-cell lung cancer (SCLC) patients has been shown to predict the disease progress and long-term survival. Most CTC detection methods rely on epithelial surface markers, such as epithelial cell adhesion molecule (EpCAM). However, this marker in SCLC is reported to be often downregulated after a variety of phenotypic changes, which impairs the reliability of EpCAM-based CTC detections. In this regard, the development of an alternative CTC detection method involving different CTC surface markers is in demand. In this study, we evaluated, for the first time to our knowledge, the feasibility of detecting SCLC CTCs using a noncatalytic endosialidase (EndoN Trap, EndoNt). This noncatalytic enzyme was chosen due to its high affinity to polysialic acid (polySia), a cell-surface glycan, that is highly expressed by SCLC tissue. Furthermore, this enzyme-based system was integrated into our dendrimer-mediated CTC capture platform to further enhance the capture efficiency via multivalent binding. We found that the EndoNt-immobilized surfaces could specifically capture polySia-positive SCLC cells and the binding between SCLC cells and EndoNt surfaces was further stabilized by dendrimer-mediated multivalent binding. When compared to the EpCAM-based capture, EndoNt significantly improved the capture efficiency of polySia-positive SCLC cells under flow due to its higher binding affinity (lower dissociation rate constants). These findings suggest that this enzyme-based CTC capture strategy has the potential to be used as a superior alternative to the commonly used EpCAM-based methods, particularly for those types of cancer that overexpress polySia.

Sulfated Alkyl Glucopyranans with Potent Antiviral Activity Synthesized by Ring-Opening Copolymerization of Anhydroglucose and Alkyl Anhydroglucose Monomers

Sulfated glucopyranans having long alkyl groups were prepared by the ring-opening copolymerization of benzylated 1,6-anhydroglucopyranose with 3-O-octadecyl 1,6-anhydro-β-d-glucopyranose monomers, and subsequent deprotection and sulfation. Water-soluble sulfated glucopyranans with 2.8 and 4.7 mol% of 3-O-octadecyl group and lower molecular weights ofM-n= 2.5 × 103–5.1 × 103have potent anti-HIV activity at 0.05–1.25 μg/mL, even though sulfated polysaccharides with molecular weights belowM-n= 6 × 103had low anti-HIV activity. The interaction with poly-l-lysine as a model compound of proteins was analyzed by SPR, DSL, and zeta potential, indicating that the sulfated 3-O-octadecyl glucopyranans had high association and low dissociation rate constants, and the particle size increased after addition of poly-l-lysine. The anti-HIV activity was induced by electrostatic interaction between sulfate groups and amino groups of poly-l-lysine and by the synergistic effect of the hydrophobic long alkyl chain and hydrophilic sulfated group.

Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents.

Limited chemical diversity of nucleic acid libraries has long been suspected to be a major constraining factor in the overall success of SELEX (Systematic Evolution of Ligands by EXponential enrichment). Despite this constraint, SELEX has enjoyed considerable success over the past quarter of a century as a result of the enormous size of starting libraries and conformational richness of nucleic acids. With judicious introduction of functional groups absent in natural nucleic acids, the “diversity gap” between nucleic acid–based ligands and protein-based ligands can be substantially bridged, to generate a new class of ligands that represent the best of both worlds. We have explored the effect of various functional groups at the 5-position of uracil and found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers). Such modified nucleotides create unique intramolecular motifs and make direct contacts with proteins. Importantly, SOMAmers engage their protein targets with surfaces that have significantly more hydrophobic character compared with conventional aptamers, thereby increasing the range of epitopes that are available for binding. These improvements have enabled us to build a collection of SOMAmers to over 3,000 human proteins encompassing major families such as growth factors, cytokines, enzymes, hormones, and receptors, with additional SOMAmers aimed at pathogen and rodent proteins. Such a large and growing collection of exquisite affinity reagents expands the scope of possible applications in diagnostics and therapeutics.

Quaternary structure effects on the hexacoordination equilibrium in rice hemoglobin rHb1: Insights from molecular dynamics simulations

Nonsymbiotic hemoglobins (nsHbs) form a widely distributed class of plant proteins, which function remains unknown. Despite the fact that class 1 plant nonsymbiotic hemoglobins are hexacoordinate (6c) heme proteins (hxHbs), their hexacoordination equilibrium constants are much lower than in hxHbs from animals or bacteria. In addition, they are characterized by having very high oxygen affinities and low oxygen dissociation rate constants. Rice hemoglobin 1 (rHb1) is a class 1 nonsymbiotic hemoglobin. It crystallizes as a fully associated homodimer with both subunits in 6c state, but showing slightly different conformations, thus leading to an asymmetric crystallographic homodimer. The residues that constitute the dimeric interface are conserved among all nsHbs, suggesting that the quaternary structure could be relevant to explain the chemical behavior and biological function of this family of proteins. In this work, we analyze the molecular basis that determine the hexacoordination equilibrium in rHb1. Our results indicate that dynamical features of the quaternary structure significantly affect the hexacoordination process. Specifically, we observe that the pentacoordinate state is stabilized in the dimer with respect to the isolated monomers. Moreover, the dimer behaves asymmetrically, in a negative cooperative scheme. The results presented in this work are fully consistent with our previous hypothesis about the key role played by the nature of the CD region in determining the coordination state of globins.

Fusion of Epstein-Barr Virus with Epithelial Cells Can Be Triggered by αvβ5 in Addition to αvβ6 and αvβ8, and Integrin Binding Triggers a Conformational Change in Glycoproteins gHgL

Fusion of herpesviruses with their target cells requires a minimum of three glycoproteins, namely, gB and a complex of gH and gL. Epstein-Barr virus (EBV) fusion with an epithelial cell requires no additional virus glycoproteins, and we have shown previously that it can be initiated by an interaction between integrin αvβ6 or αvβ8 and gHgL. We now report that integrin αvβ5 can also bind to gHgL and trigger fusion. Binding of gHgL to integrins is a two-step reaction. The first step, analyzed by surface plasmon resonance, was fast, with high association and low dissociation rate constants. The second step, detected by fluorescence spectroscopy of gHgL labeled at cysteine 153 at the domain I-domain II interface with the environmentally sensitive probes acrylodan and IANBD, involved a slower conformational change. Interaction of gHgL with neutralizing monoclonal antibodies or Fab' fragments was also consistent with a two-step reaction involving fast high-affinity binding and a subsequent slower conformational change. None of the antibodies bound to the same epitope, and none completely inhibited integrin binding. However, binding of each decreased the rate of conformational change induced by integrin binding, suggesting that neutralization might involve a conformational change that precludes fusion. Overall, the data are consistent with the interaction of gHgL with an integrin inducing a functionally important rearrangement at the domain I-domain II interface.

Direct Detection of Thrombin Binding to 8-Bromodeoxyguanosine-Modified Aptamer: Effects of Modification on Affinity and Kinetics

The affinity of an 8-bromodeoxyguanosine- (8-BrdG-) substituted thrombin-binding aptamer (TBA-Br), which has the 1st and 10th guanosine residues replaced with 8-BrdG, was estimated using reflectometric interference spectroscopy (RIfS). When comparing TBA-Br with unmodified TBA (TBA-H), it was demonstrated that the modification effectively improved the affinity of TBA; dissociation constants (KD) of TBA-H and TBA-Br were 45.4 nM and 1.99 nM, respectively. These values, which were obtained by direct observation of thrombin binding using RIfS, have the same order of magnitude as those obtained in our previous study utilizing conformational changes in TBA to detect thrombin binding, thus confirming the validity of the obtained KD values. RIfS measurements also revealed that the 8-BrdG modification resulted in a lower dissociation rate constant (kd), which suggests that the enhancement of affinity can be attributed to the stabilization of the G-quadruplex structure on introduction of 8-BrdG.

Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant ( k off). Since no techniques are available to estimate k off, we report herewith a method to determine k off based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k off value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)–cyanide) was determined at pH 9.2 and 20.0 °C. Mixing Mb(III)–cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)–cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)–cyanide dissociation, the k off value, representing the rate-limiting step, can be directly determined. The k off value obtained experimentally matches very well to that calculated from values of the second-order rate constant ( k on) and of the dissociation equilibrium constant ( K) for cyanide binding to Mb(III) ( k off = k on × K).

Kinetic Analysis of the Interactions Between the Different ELL and EAF Proteins Involved in Acute Myeloid Leukemia

The ELL gene was first cloned as a fusion partner of MLL in the (11;19)(q23;p13.1) translocation that occurs in acute myeloid leukemia. Subsequently, the ELL2 gene was cloned on the basis of its sequence homology to ELL. Both proteins stimulate the rate of transcript elongation by RNA polymerase II. Previously, we isolated two closely related proteins, EAF1 and EAF2, which interact with ELL and ELL2. Deletion mapping studies carried out to delineate the domain(s) of ELL involved in its interaction with either EAF1 or EAF2 showed that the N-terminus (amino acids 1–207) of ELL binds to both EAF1 and EAF2. In comparison, the middle region (207–411 amino acids) does not bind to either of the two EAF proteins and the C-terminus region (411–621 amino acids) binds only to the EAF1 protein. Biochemical studies have revealed that EAF1 and EAF2 enhance the rate of mRNA chain elongation by the ELL proteins in vitro. Although both ELL and ELL2 have similar roles in transcriptional elongation, ELL2 has not been shown to be involved in any hematological abnormality so far. In an attempt to gain a deeper understanding of the biology and functions of the interactions between these different proteins, we determined the kinetic properties of these interactions using the biophysical techniques of surface plasmon resonance (SPR) and isothermal calorimetry (ITC). SPR detects complex formation in real time and provides a better comprehension of the dynamics of association and dissociation of an interaction, and ITC is used to determine the thermodynamics of the interaction. Our SPR analysis has provided novel insights into the nature of the binding of the ELL proteins to the EAF proteins. We observed that both ELL and ELL2 bind to EAF1 and EAF2 with a high affinity, but the binding affinity of ELL2 for both EAF1 and EAF2 is almost twelve-fold greater than the affinity of ELL for both the EAF proteins. The higher affinity of ELL2 is due to much slower uptake and release kinetics reflected by the low association and dissociation rate constants of ELL2 compared to ELL. The stoichiometry of ELL, ELL2, EAF1 and EAF2 in the ELL-EAF1, ELL-EAF2, ELL2-EAF1 and ELL2-EAF2 complexes was estimated to be 1:1 after fitting the respective sensorgrams obtained by SPR analysis to the Langmuir's bimolecular model. Interestingly, we did not observe any difference in the affinity of either ELL or ELL2 for binding to EAF1 or EAF2. We used SPR-based competition experiments to show that ELL and ELL2 bind to the same sites on the EAF proteins. We have also investigated the characteristics of binding of the various ELL domains to the EAF1/2 proteins. In the (11;19)(q23;p13.1) translocation, the C-terminus of ELL fuses to the N-terminus of MLL to generate a chimeric protein that interacts with EAF1 and this interaction is critical for the role of ELL in cell immortalization in vitro and leukemogenesis in vivo. In agreement with this observation, we found that the C-terminus of ELL binds EAF1 with a higher affinity than EAF2, while the N-terminus of ELL binds with similar affinities and displays similar kinetics of binding to both EAF1 and EAF2. We also found that the individual binding sites on the ELL N-terminus and the C-terminus exhibited a lower affinity for the EAF proteins, but the affinity increases when the two sites function together in the context of the full-length protein, suggesting that the two sites co-operate with each other to increase the affinity for the full-length ELL protein. Taken together, these observations suggest that although ELL and ELL2 share many similarities in terms of their sequence and function in transcription elongation, they bind to the EAF proteins with different affinities and kinetics. Alternative interaction dynamics and the interplay between the different ELL and EAF proteins permit distinct functional regulation of transcriptional elongation in normal and leukemic cells.

Epigallocatechin gallate is a slow-tight binding inhibitor of enoyl-ACP reductase from Plasmodium falciparum

Epigallocatechin gallate (EGCG) is known to have numerous pharmacological properties. In the present study, we have shown that EGCG inhibits enoyl–acyl carrier protein reductase of Plasmodium falciparum (PfENR) by following a two-step, slow, tight-binding inhibition mechanism. The association/isomerization rate constant (k5) of the reversible and loose PfENR–EGCG binary complex to a tight [PfENR–EGCG]∗ or EI∗ complex was calculated to be 4.0×10−2s−1. The low dissociation rate constant (k6) of the [PfENR–EGCG]∗ complex confirms the tight-binding nature of EGCG. EGCG inhibited PfENR with the overall inhibition constant (Ki∗) of 7.0±0.8nM. Further, we also studied the effect of triclosan on the inhibitory activity of EGCG. Triclosan lowered the k6 of the EI∗ complex by 100 times, lowering the overall Ki∗ of EGCG to 97.5±12.5 pM. The results support EGCG as a promising candidate for the development of tea catechin based antimalarial drugs.

Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1

The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 nM for the IgG and 10 μM for the Fab), with particularly low but similar dissociation rate constants (0.031–0.095 s−1). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.

A Study on p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Conventional methods to prepare the apoenzyme of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens yield an inhomogeneous apoenzyme with respect to the reconstitutable activity. To overcome this problem the holoenzyme was bound covalently to a Sepharose–5,5′-dithio-bis(2-nitrobenzoate) column via the reactive sulfhydryl group of the enzyme. The prosthetic group FAD was removed at neutral pH by addition of a high concentration of KBr and urea to the elution buffer. The holoenzyme can be reconstituted while the apoenzyme is still bound to the column or the apoenzyme can be isolated in the free state. The yield of apoenzyme was between 90% and 95% of the starting enzyme. The degree of reconstitution of holoenzyme is better than 95% of the original activity. The apoenzyme is stable for a long period of time as an ammonium sulfate precipitate. The relative molecular mass of the apoenzyme is 43000 per polypeptide chain. The apoenzyme exists mainly as a dimer in solution. During the preparation of the apoenzyme the substrate p-hydroxybenzoate protects the apoenzyme from inactivation, indicating complex formation. The dissociation constant for the apoenzyme-substrate complex was determined to be about 30 μM. The apoenzyme also forms a complex with NADPH. The dissociation constant (∼ 12 μM) of this complex is about a factor of ten smaller than that of the holoenzyme (∼ 120 μM). The dissociation constant of the apoenzyme-NADPH complex is strongly dependent on pH and ionic strength of the solution. The dissociation constant of the holoenzyme into its constituents is 45 nM. The association rate constant calculated from kinetic experiments is 4.1 × 104 M−1 s−1. The calculated dissociation rate constant is 1.85 × 10−3 s−1, indicating that the holoenzyme possesses a high stability which is governed by the low dissociation rate constant. The method of preparation of apoenzyme can also be used to separate enzyme molecules where the reactive SH group has been oxidized by air from enzyme molecules still containing the original SH group. This separation technique is in particular useful when quantitative labelling of the SH group is required.

Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases.

Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.

Identification of RNA ligands that bind hepatitis C virus polymerase selectively and inhibit its RNA synthesis from the natural viral RNA templates

To identify the potential RNA inhibitors of HCV polymerase, we have isolated high-affinity RNA ligands specific to hepatitis C virus (HCV) NS5B protein from a combinatorial RNA library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) procedure. Thirty-seven selected ligands were classified into eight groups on the basis of their sequence homologies. Most (60%) of the ligands carry the conserved YGUAGR hexamer (Y = pyrimidine, R = purine) at the 5′ end of the 40-nt randomized region, and 74% of the ligands end in (A/C)U at the 3′end. However, strong binding to NS5B required the whole RNA ligand including the flanking conserved nucleotides at both ends. The binding of the selected ligands to NS5B is highly specific and strong, as reflected in their low dissociation rate constants ( k d ∼ 10 −4 s −1). Analysis of secondary structure by computer program and RNase footprints of the two different aptamers from two most conserved groups revealed RNA structures containing three stem loops with internal bulges. NS5B bound these RNA at a region between the two stem loops from the 5′ -end. Some of these RNA aptamers could serve as a template for the HCV polymerase, but some interfered with the activity of the viral enzyme. These RNA ligands will be useful for further characterization of NS5B-binding properties and, with further modifications, may have potential therapeutic value.

Controlling Ligand Binding in Myoglobin by Mutagenesis

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.

Concentration jump experiments for the precise determination of rate constants of reverse reactions in the millisecond time range.

Low dissociation or reverse rate constants of single-step or multistep complex formation equilibria are usually obtained with reduced precision from standard stopped-flow binding experiments by determination of the intercept of the concentration dependence of k(obs). Large and fast concentration jumps, based on two different step-motor-driven mixing setups, are performed with 60-300-fold dilutions that allow the precise, convenient, and independent determination of dissociation rate constants in the range of approximately 0.1-100 s(-1) in a single stopped-flow dissociation experiment. A theoretical basis is developed for the design and for the evaluation of such dilution experiments by considering the rebinding occurring during dissociation. The kinetics of three chemical systems are investigated, the binding of Mg2+ to 8-hydroxyquinoline as well as of Ca2+ and K+ to the cryptand [2.2.2], by carrying out standard stopped-flow binding as well as dissociation experiments employing various dilution factors. The advantage of the dilution method for investigating chemical and biological systems is emphasized.

Interaction between the Amino-terminal SH3 Domain of CRK and Its Natural Target Proteins

CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), Shc, and paxillin. The SH3 domain, in turn, binds to cytosolic proteins of 135-145, 160, 180, and 220 kDa. We screened expression libraries by Far Western blotting, using CRK SH3 as a probe, and identified partial cDNA sequences of four distinct proteins, including C3G, DOCK180, EPS15, and clone ST12. The consensus sequence of the CRK SH3 binding sites as deduced from their amino acid sequences was Pro+3-Pro+2-X+1-Leu0-Pro-1-X-2-Lys-3. The interaction of the CRK SH3 domain with the DOCK180 peptide was examined with an optical biosensor, based on the principles of surface plasmon resonance. A low dissociation constant of the order of 10(-7) resulted from a high association rate constant (kassoc = 3 x 10(4)) and low dissociation rate constant (kdiss = 3 x 10(-3)). All CRK-binding proteins except clone ST12 also bound to another adaptor protein, Grb2. Mutational analysis revealed that glycine at position +1 of ST12 inhibited the binding to Grb2 while retaining the high affinity binding to CRK SH3. The result suggests that the amino acid at position +1 also contributes to the high affinity binding of the peptides to the SH3 domain of Grb2, but not to that of CRK.

Selection of phage displayed antibodies based on kinetic constants

The display of antibody fragments on the surface of filamentous bacteriophages and the selection of binders from antibody libraries have provided powerful tools to generate human antibodies. We reported recently a new concept (SAP system) for the selection of specific phages by linking antigenic recognition and phage replication, using a soluble fusion protein containing the antigen and a fragment of the M13 coat protein 3. In this investigation, a model library has been composed using six different antibody fragments which were characterized individually regarding their k ass, k diss and K a. All Fab fragments were specific for a 15 amino acid region of the V3 loop of gp120 (HIV-1). We demonstrated that the SAP system could discriminate between the kinetic parameters of each clone, using different selection strategies. Phages expressing high affinity clones were selected preferentially using low doses of antigen but clones of lower affinity also could be selected by increasing the antigen concentration or using a preselection procedure. Phages expressing antibody fragment with high association or low dissociation rate constants were retrieved by utilizing short contact times between antigen and antibody or antigen-chase conditions.

Identification of functional and structural amino-acid residues by parsimonious mutagenesis

For in vitro evolution of protein function, we previously proposed using parsimonious mutagenesis (PM), a technique where mutagenic oligodeoxynucleotides (oligo) are designed to minimize coding sequence redundancy and limit the number of amino acid (aa) residues which do not retain parental structural features. For this work, PM was used to increase the affinity of C6.5, a human single-chain Fv (scFv) that binds the glycoprotein tumor antigen, c-erbB-2. A phage antibody library was created where 19 aa located in three of the heavy (H) and light (L) chain antigen-binding loops (L1, L3 and H2) were simultaneously mutated. After four rounds of selection, 50% of scFv had a lower dissociation rate constant ( koff) than the parental scFv. The K d of these scFv ranged from twofold ( K d=7.0 × 10 9M) to sixfold ( K d =2.4 × 10 9M) lower than the parental scFv ( K d = 1.6 × 10 −8M). In higher affinity scFv, substitutions occurred at 10 19 of the positions, with 21 28 substitutions occurring at only four positions, two in H2, and one each in L1 and L3. Only the wild type (wt) aa was observed at 9 19 aa. Based on a model of C6.5, seven of the nine conserved aa have a structural role in the variable domain, either in maintaining the main chain conformation of the loop, or in packing on the H-chain variable domain. Two of the conserved aa are solvent exposed, suggesting they may play a critical role in recognition. Thus, PM identified three types of aa: structural aa, functional aa which modulate affinity, and functional aa, which are critical for recognition. Since the sequence space was not completely sampled, higher affinity scFv could be produced by subjecting functional aa which modulate affinity to a higher rate of mutation. Furthermore, PM could prove useful for modifying function in other proteins that belong to structurally related families