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To initiate infection of Escherichia coli, phage fd uses its gene-3-protein (G3P) to bind first to an F pilus and then to the TolA protein at the cell surface. G3P is normally auto-inhibited because a tight interaction between the two N-terminal domains N1 and N2 buries the TolA binding site. Binding of N2 to the pilus activates G3P by initiating long-range conformational changes that are relayed to the domain interface and to a proline timer. We discovered that the 23-28 loop of the N1 domain is critical for propagating these conformational signals. The analysis of the stability and the folding dynamics of G3P variants with a shortened loop combined with TolA interaction studies and phage infection experiments reveal how the contact between the N2 domain and the 23-28 loop of N1 is energetically linked with the interdomain region and the proline timer and how it affects phage infectivity. Our results illustrate how conformational transitions and prolyl cis/trans isomerization can be coupled energetically and how conformational signals to and from prolines can be propagated over long distances in proteins.

The fd bacteriophage is a filamentous virus consisting of a circular single-stranded DNA (ssDNA) wrapped by thousands of copies of a major coat protein subunit (the capsid). The coat protein subunits are mostly α-helical and curved, and are arranged in the capsid in consecutive pentamers related by a translation along the main viral axis and a rotation of ~36° (C5S2 symmetry). The DNA is right-handed and helical, but information on its structure and on its interface with the capsid is incomplete. We present here an approach for assigning the DNA nucleotides and studying its interactions with the capsid by magic-angle spinning solid-state NMR. Capsid contacts with the ssDNA are obtained using a two-dimensional (13)C-(13)C correlation experiment and a proton-mediated (31)P-(13)C polarization transfer experiment, both acquired on an aromatic-unlabeled phage sample. Our results allow us to map the residues that face the interior of the capsid and to show that the ssDNA-capsid interactions are sustained mainly by electrostatic interactions between the positively charged lysine side chains and the phosphate backbone. The use of natural abundance aromatic amino acids in the growth media facilitated the complete assignment of the four nucleotides and the observation of internucleotide contacts. Using chemical shift analysis, our study shows that structural features of the deoxyribose carbons reporting on the sugar pucker are strikingly similar to those observed recently for the Pf1 phage. However, the ssDNA-protein interface is different, and chemical shift markers of base pairing are different. This experimental approach can be utilized in other filamentous and icosahedral bacteriophages, and also in other biomolecular complexes involving structurally and functionally important DNA-protein interactions.

Major histocompatibility complex (MHC) molecules bind peptides bearing an appropriate 'sequence motif' for MHC binding. The use of phage display libraries exploits the ability of MHC class II molecules to exchange peptides in solution and thus select out peptide sequences with high-affinity binding from a large array of random peptides. We have analysed the peptide binding motifs of HLA-DRB1*1301 and *1302 using affinity purified HLA-DR13 molecules to purify sequentially HLA-DR13-binding peptides from a large random library of M13 phage containing nonamer inserts in the pIII coat protein. These DR13 alleles differ only at position 86 of the HLA-DR beta chain, where they contain valine and glycine residues respectively. These alleles were chosen because of their association with protection from severe malaria and chronic hepatitis B virus infection in West Africa. Analysis of the phage bound to these DR molecules suggests binding motifs. We compare the results derived from the use of the phage display library with results obtained from analysis of eluted peptides and peptide-binding studies. This analysis shows that although there is a common theme to motifs derived using different methods, there are also subtle variations between them.

This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve diversity of phage clones and identify ligands previously lost in phage display screens.

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C ≫ A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA−Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II. Mutation spectrum established for strains expressing only Pol V, showed that in uvrA− bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T → C:G, A:T → G:C, G:C → A:T and G:C → T:A prevailed. The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.

CD44, a cancer-associated membrane glycoprotein involved in cell adhesion and tumor progression, has been implicated as a cancer stem cell antigen in several cancers including breast cancer. If the detection sensitivity of CD44 as an early marker for cancer could be improved, this would have important clinical applications. As compared with early stage treatments of other kinds of cancer, treatment of breast cancer is more likely to results in positive outcomes, so this early detection is crucial. Therefore, CD44 is a potential diagnostic target for cancer detection. Herein, we have used a peptide library to screen novel diverse peptides that bind to CD44 with high affinity and characterized the specific binding of these peptides. Our work provides a basis to develop novel diagnostic peptides which may replace antibodies as CD44 detection probes.

In Alzheimer's disease (AD), amyloid-β (Aβ) peptides accumulate in the brain in different forms, including fibrils and oligomers. Recently, we established three distinct conformation-dependent human single-chain Fv (scFv) antibodies, including B6 scFv, which bound to Aβ42 fibril but not to soluble-form Aβ, inhibiting Aβ42 fibril formation. In this study, we determined the mimotopes of these antibodies and found a common mimotope sequence, B6-C15, using the Ph.D.-C7C phage library. The B6-C15 showed weak homology to the C-terminus of Aβ42 containing GXXXG dimerization motifs. We synthesized the peptide of B6-C15 fused with biotinylated TAT at the N-terminus (TAT-B6-C15) and characterized its biochemical features on an Aβ42-fibrillation reaction in vitro. We demonstrated that, first, TAT-B6-C15 inhibited Aβ42 fibril formation; secondly, TAT-B6-C15 bound to prefibril Aβ42 oligomers but not to monomers, trimers, tetramers, fibrils, or ultrasonicated fragments; thirdly, TAT-B6-C15 inhibited Aβ42-induced cytotoxicity against human SH-SY5Y neuroblastoma cells; and, fourthly, when mice were administered B6-C15-phages dissolved in phosphate-buffered saline, the anti-Aβ42 conformer IgG antibody response was induced. These results suggested that the B6-C15 peptide might provide unique opportunities to analyze the Aβ42 fibrillation pathway and develop a vaccine vehicle for Alzheimer's disease.

The filamentous phage fd uses its gene 3 protein (G3P) to target Escherichia coli cells in a two-step process. First, the N2 domain of G3P attaches to an F pilus, and then the N1 domain binds to TolA-C. N1 and N2 are tightly associated, rendering the phage robust but noninfectious because the binding site for TolA-C is buried at the domain interface. Binding of N2 to the F pilus initiates partial unfolding, domain disassembly, and prolyl cis-to-trans isomerization in the hinge between N1 and N2. This activates the phage, and trans-Pro213 maintains this state long enough for N1 to reach TolA-C. Phage IF1 targets I pili, and its G3P contains also an N1 domain and an N2 domain. The pilus-binding N2 domains of the phages IF1 and fd are unrelated, and the N1 domains share a 31% sequence identity. We show that N2 of phage IF1 mediates binding to the I pilus, and that N1 targets TolA. Crystallographic and NMR analyses of the complex between N1 and TolA-C indicate that phage IF1 interacts with the same site on TolA-C as phage fd. In IF1-G3P, N1 and N2 are independently folding units, however, and the TolA binding site on N1 is permanently accessible. Activation by unfolding and prolyl isomerization, as in the case of phage fd, is not observed. In IF1-G3P, the absence of stabilizing domain interactions is compensated for by a strong increase in the stabilities of the individual domains. Apparently, these closely related filamentous phages evolved different mechanisms to reconcile robustness with high infectivity.

Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment. Assembly of functional Fab was confirmed by demonstration of antigen-specific binding using antibodies of known specificity. Phage displaying a Fab specific for RSV-F protein with Fd linked to pIX showed efficient, antigen-specific enrichment when mixed with phage displaying a different specificity. The functionality of this system for antibody engineering was evaluated in an optimization study. A RSV-F protein specific antibody with an affinity of about 2nM was randomized at 4 positions in light chain CDR1. Three rounds of selection with decreasing antigen concentration yielded Fabs with an affinity improvement up to 70-fold and showed a general correlation between enrichment frequency and affinity. We conclude that the pIX coat protein complements other display systems in filamentous phage as an efficient vehicle for low copy display and selection of Fab proteins.

Y-family DNA polymerases (DNAPs) are often required in cells to synthesize past DNA-containing lesions, such as [+ta]-B[a]P-N(2)-dG, which is the major adduct of the potent mutagen/carcinogen benzo[a]pyrene. The current model for the non-mutagenic pathway in Escherichia coli involves DNAP IV inserting deoxycytidine triphosphate opposite [+ta]-B[a]P-N(2)-dG and DNAP V doing the next step(s), extension. We are investigating what structural differences in these related Y-family DNAPs dictate their functional differences. X-ray structures of Y-family DNAPs reveal a number of interesting features in the vicinity of the active site, including (1) the "roof-amino acid" (roof-aa), which is the amino acid that lies above the nucleobase of the deoxynucleotide triphosphate (dNTP) and is expected to play a role in dNTP insertion efficiency, and (2) a cluster of three amino acids, including the roof-aa, which anchors the base of a loop, whose detailed structure dictates several important mechanistic functions. Since no X-ray structures existed for UmuC (the polymerase subunit of DNAP V) or DNAP IV, we previously built molecular models. Herein, we test the accuracy of our UmuC(V) model by investigating how amino acid replacement mutants affect lesion bypass efficiency. A ssM13 vector containing a single [+ta]-B[a]P-N(2)-dG is transformed into E. coli carrying mutations at I38, which is the roof-aa in our UmuC(V) model, and output progeny vector yield is monitored as a measure of the relative efficiency of the non-mutagenic pathway. Findings show that (1) the roof-aa is almost certainly I38, whose beta-carbon branching R-group is key for optimal activity, and (2) I38/A39/V29 form a hydrophobic cluster that anchors an important mechanistic loop, aa29-39. In addition, bypass efficiency is significantly lower both for the I38A mutation of the roof-aa and for the adjacent A39T mutation; however, the I38A/A39T double mutant is almost as active as wild-type UmuC(V), which probably reflects the following. Y-family DNAPs fall into several classes with respect to the [roof-aa/next amino acid]: one class has [isoleucine/alanine] and includes UmuC(V) and DNAP eta (from many species), while the second class has [alanine (or serine)/threonine] and includes DNAP IV, DNAP kappa (from many species), and Dpo4. Thus, the high activity of the I38A/A39T double mutant probably arises because UmuC(V) was converted from the V/eta class to the IV/kappa class with respect to the [roof-aa/next amino acid]. Structural and mechanistic aspects of these two classes of Y-family DNAPs are discussed.

The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped alpha-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein. Recently, site-directed labelling approaches have enabled the study of the coat protein under conditions that more closely mimic the in vivo membrane-bound state. Interestingly, the structure that has emerged from this work is I-shaped and similar to the structure in the phage-bound state.

An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pI proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2+/-0.7 x 10(5)M(-1) which indicates a strong binding close to that of antibody.

Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two curved approximately 200 basepair (bp) restriction fragments, one taken from the M13 origin of replication and the other from the VP1 gene of SV40. These two fragments have previously been shown to contain stable, centrally located bends of 44 degrees and 46 degrees , respectively, located within approximately 60 bp "curvature modules" containing four or five irregularly spaced A-tracts. Transient electric birefringence measurements of these two fragments, sequence variants containing reduced numbers of A-tracts in the SV40 curvature module or changes in the residues flanking the A-tracts in the M13 curvature module, have been combined with the free solution electrophoretic mobilities of the same fragments using known equations to estimate the effective charge of each fragment. The effective charge is reduced, on average, by one-third charge for each A-tract in the curvature module, suggesting that each A-tract binds a monovalent cation approximately one-third of the time. Monovalent cation binding to two or more A-tracts is required to observe significant curvature of the DNA helix axis.

Topogenic sequences direct the membrane topology of proteins by being recognized and decoded by integral membrane translocases. In this paper, we have compared the minimal sequence characteristics of helical-hairpin, reverse signal-anchor, and stop-transfer sequences in bacterial membrane proteins that use either the YidC or SecYEG translocases for membrane insertion. We find that a stretch composed of 3 leucines and 16 alanines is required for efficient membrane-anchoring of the M13 procoat protein that inserts by a helical hairpin mechanism, and that a stretch composed of only 19 alanines has a detectable membrane-anchoring ability. Similar results were obtained for the reverse signal-anchor sequence of the single-spanning Pf3 coat protein and for stop-transfer segments engineered into leader peptidase. We have also determined the contribution to the apparent free energy of membrane insertion of M13 procoat for all 20 amino acids. The relative order of the contributions is similar to that determined for a stop-transfer sequence in the mammalian endoplasmic reticulum, but the absolute difference between the contributions for the most hydrophobic and most hydrophilic residues is somewhat larger in the E. coli system. These results are significant because they define the features of a membrane protein transmembrane segment that induce lateral release from the YidC and Sec translocases into the lipid bilayer in bacteria.

A single-chain variable fragment (scFv) antibody library against Bordetella pertussis was constructed using M13 phage display. The library was enriched for phages surface displaying functional scFv by biopanning against B. pertussis immobilized on polystyrene plates. Two hundred eighty-eight individual clones from the enriched library were screened for binding to B. pertussis cells, filamentous hemagglutinin (FHA), and pertactin (PRN) in enzyme-linked immunosorbent assays (ELISAs). Based on the binding ability, the clones were put into eight groups. The scFv DNA inserts from the 288 clones were digested with BstOI, and 18 unique restriction patterns, named types 1 to 18, were found. Eight clones (types 1 to 7 and 18) were selected for further testing against FHA, PRN, and B. pertussis by ELISA. The results showed that types 1, 5, 7, and 18 bound strongly to B. pertussis cells as well as FHA and PRN. Type 3 bound strongly to the cells and FHA but weakly to PRN. Types 4 and 6 bound FHA only, and type 2 did not bind to the cells or antigens. The ability of the eight clones to inhibit B. pertussis from binding to HEp-2 cells was assayed. Types 1, 5, and 7, but not the remaining clones, inhibited the adherence of B. pertussis to HEp-2 cells. The scFvs were sequenced, and the deduced amino acid sequence showed that the scFvs were different antibodies. Maltose-binding protein (MBP) fusion proteins composed of three different regions of FHA (heparin-binding domain, carbohydrate recognition domain, and the RGD triplet motif) were constructed. The three fusion proteins and Mal85 (MBP-FHA type I domain) were used to map the binding sites for scFvs of types 1, 5, and 7 by ELISA. The results showed that all three scFvs bound to the heparin-binding domain fusion protein but not the other fusion proteins. BALB/c mice who received recombinant phage-treated B. pertussis had reduced bacterial counts in the nasal cavity, trachea, and lungs compared to the control groups.

Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function.

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.

We have developed a system for directly isolating foreign peptides displayed on the N-terminus of the major coat protein of bacteriophage M13. The phage particle in this system is formed as a mixture of wild type and modified coat proteins. The N-terminal segment of the modified coat protein was mutated for chemical cleavage, in order to obtain the displayed peptide from the major coat protein. Using 13C, 15N- labeled medium, we introduced stable isotopes, 13C and/or 15N, into the coat proteins. The NMR spectra for the cleaved peptides from the phage particles could be recorded within a few days after the selection of the phage clone.

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12-130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7x10(4)-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffman's n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18-24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region.

Streptavidin, a homotetrameric protein with extremely tight biotin binding ( K d ≤ 10 -14 M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. K d ′ values for B4F ranged from ≈10 -11 to 10 -10 M, although K d values for biotin ranged from 10 -6 to 10 -5 M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin–biotin for composite separation or analysis steps.

New insights into the low-resolution Structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process.

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

DNA repair via the homologous recombination pathway requires the recombinase RAD51 and, in vertabrates, five RAD51 paralogs. The paralogs form two complexes in solution, a XRCC3/RAD51C heterodimer and a RAD51B/RAD51C/RAD51D/XRCC2 heterotetramer. Mutation of any one of the five paralog genes prevents subnuclear assembly of recombinase at damaged sites and renders cells 30–100 fold sensitive to DNA cross-linking drugs. Phage display was used to isolate peptides that bind the paralog XRCC3. Sequences of binding peptides showed similarity to residues 14–25 of RAD51C protein. Point mutations in this region of RAD51C altered its interaction with both XRCC3 and RAD51B in a two-hybrid system. A synthetic peptide composed of residues 14–25 of RAD51C fused to a membrane transduction sequence [protein transduction domain 4 (PTD4)], inhibited subnuclear assembly of RAD51 recombinase, and sensitized Chinese hamster ovary cells to cisplatin when added to growth medium. These results suggest that residues 14–25 of RAD51C contribute to a “hot spot” used in both XRCC3-RAD51C and RAD51B-RAD51C interactions. Peptide-based inhibition of homologous recombination may prove useful for improving the efficacy of existing cancer therapies.

Here, we reported a conformation-constrained peptide library, that was constructed based on the scaffold of a 29 amino acids peptide derived from insect defensin A. The peptide scaffold was designed utilizing the InsightII molecular modeling software and then displayed on M13 filamentous bacteriophage by fusion with coat protein III. The library was constructed by randomization of seven positions located within the two loops of the peptide scaffold generating approximately 8.3 x 10(8) transformants. Sequences from 14 randomly selected phage clones indicated that the distribution of nucleotides and amino acids paralleled with the expected frequency. Screening against the target proteins: tumor necrosis factor alpha, TNF receptor 1, TNF receptor 2 and monoclonal antibody against BMP-2 showed significant enrichment in all cases. The results presented here show that the reconstructed insect defensin A domain will be a promising non-antibody protein scaffold for the presentation of a phage-displayed constrained peptide library.

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787–817 and 841–869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn858, Glu836, and Trp799 reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.

A library of constrained peptides that form stable folded structures was screened for aptamers that bind with high affinity to the fluorescent dye Texas red. Two selected clones had binding constants to Texas red of 25 and 80 pM as phage and binding had minimal effects on the fluorescence of Texas red. The peptides interact with distinct but overlapping regions of Texas red. One peptide bound to X-rhod calcium sensors, which share the same core fluorophore as Texas red. These dyes retained calcium sensitivity when bound to the peptide. This peptide was used to label a fusion protein with X-rhod-5F in vivo, and X-rhod sensed changes in calcium locally. Thus, minimal, constrained peptides can functionally bind to environmentally sensitive dyes or other organic agents in biological contexts, suggesting tools for in vivo imaging and analysis.

CD47, a cell surface transmembrane Ig superfamily member, is an extracellular ligand for signal regulatory protein (SIRPα). Interactions between CD47 and SIRPα regulate many important immune cell functions including neutrophil (PMN) transmigration. Here we report identification of a novel function-blocking peptide, CERVIGTGWVRC, that structurally mimics an epitope on CD47 and binds to SIRPα. The CERVIGTGWVRC sequence was identified by panning phage display libraries on the inhibitory CD47 mAb, C5D5. In vitro PMN migration assays demonstrated that peptide CERVIGTGWVRC specifically inhibited PMN migration across intestinal epithelial monolayers and matrix in a dose-dependent fashion. Further studies using recombinant proteins indicated that the peptide specifically blocks CD47 and SIRPα binding in a dose-dependent fashion. Protein binding assays using SIRPα domain-specific recombinant proteins demonstrated that this peptide directly bound to the distal-most Ig loop of SIRPα, the same loop where CD47 binds. In summary, these findings support the relevance of CD47-SIRPα interactions in regulation of PMN transmigration and provide structural data predicting the key residues involved on the surface of CD47. Such peptide reagents may be useful for studies on experimental models of inflammation and provide a template for the design of anti-inflammatory agents.

We report the development of a system for displaying bivalent antibody fragments on M13 bacteriophage in a manner that effectively mimics the binding behavior of natural antibodies. In the "bivalent display" format, two copies of antigen binding sites are displayed on the coat of a single phage particle. Bivalent display was first achieved by the insertion of a dimerization domain, consisting of an IgG1 hinge region and a homodimerizing GCN4 leucine zipper, between a Fab and the C-terminal domain of the M13 gene-3 minor coat protein. In a phagemid-based display system, the resulting "Fab'-zip-phage" particles display bivalent Fabs that resemble natural IgGs. An important functional consequence of bivalent display is an avidity effect, which results in a greatly reduced off-rate for phage bound to immobilized antigen. The avidity effect improved the capture and retention of bivalent Fab'-zip-phage relative to monovalent Fab-phage both with antigen immobilized on plates and with cell surface antigen. To examine the requirements for bivalent display on phage, we systematically trimmed down the dimerization domain and found that a single cysteine was sufficient to confer the same avidity effect conferred by the complete dimerization domain. Bivalent antibody phage display should be useful for many applications. In particular, the technology should aid in the production of antibodies against difficult antigens, and also, in selections that require dimerization for activity.

A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display. This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.

The dramatic improvement in the NMR spectra of insulin-like growth factor I (IGF-I) in the presence of a peptide identified from a phage display library has allowed for the first time the determination of a high-resolution solution structure for much of IGF-I. The three helices of IGF-I in this complex have an arrangement similar to that seen in high-resolution crystal structures of IGF-I and insulin, although there are differences in the conformation and precise location of helix 3. A cluster of hydrophobic and basic side chains within the turn-helix motif of the peptide contact a hydrophobic patch on helices 1 and 3 of IGF-I. The importance of this patch for tight binding was verified using alanine scanning mutagenesis of the peptide in two different phage display formats. Consistent with its antagonistic activity, the peptide binds to a region implicated by mutagenesis studies to be important for association with IGF binding proteins (IGFBPs). The ability of the peptide to also inhibit signaling has important implications for the manner in which IGF-I interacts with its receptor. Interestingly, the peptide uses the same binding site as detergent and a fragment of IGFBP-5 identified in other IGF-I complexes. The ligand-induced structural variability of helix 3 in these complexes suggests that exchange between such conformations may be the source of the dynamic nature of free IGF-I and likely has functional significance for the ability of IGF-I to recognize two signaling receptors and six binding proteins with high affinity.

CTXφ is a filamentous bacteriophage that encodes cholera toxin. CTXφ infection of its host bacterium, Vibrio cholerae , requires the toxin-coregulated pilus (TCP) and the products of the V. cholerae tolQRA genes. Here, we have explored the role of OrfU, a predicted CTXφ minor coat protein, in CTXφ infection. Prior to the discovery that it was part of a prophage, orfU was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences. Based on its size and position in the CTXφ genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXφ infection as well as playing a role in CTXφ assembly and release. Deletion of orfU from CTXφ dramatically reduced the number of CTXφ virions detected in supernatants from CTXφ-bearing cells. This defect was complemented by expression of orfU in trans , thereby confirming a role for this gene in CTXφ assembly and/or release. To evaluate the requirement for OrfU in CTXφ infection, we introduced fragments of orfU into gIII in an fd derivative to create OrfU-pIII fusions. While fd is ordinarily unable to infect V. cholerae , an fd phage displaying the N-terminal 274 amino acids of OrfU could infect V. cholerae in a TCP- and TolA-dependent fashion. Since our findings indicate that OrfU functions as the CTXφ pIII, we propose to rename OrfU as pIII CTX . Our data also provide new evidence for a conserved pathway for filamentous phage infection.

Expression of single-chain variable fragment antibody on the surface of filamentous bacteriophage is widely used to make antibodies with pre-defined specificities. Using direct selection on solid phase-bound amyloid-beta 40 peptide, which is a main pathogenic feature of Alzheimer's disease, we obtained single-chain Fv antibody with specific binding activity. The binding epitope of the antibody is located between amino acids 1 and 16 of the peptide antigen. DNA sequencing showed that the gene coding for the single chain Fv antibody consists of 768 bp, and the complementarity-determining regions were deduced.

A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli. The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP ( K(m)=34 micro mu) and a divalent cation (Mn(2+), Mg(2+), or Ca(2+)). dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD(+) were inactive. The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn(2+) than in the presence of Mg(2+) or Ca(2+). Unexpectedly, the native Ssh ligase preferred Mg(2+) and Ca(2+) rather than Mn(2+). Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80 degrees C. Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end. The enzyme showed little activity on a 1-nucleotide gapped substrate. This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3′ exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol+ Exo+) enzyme, an exonuclease-deficient mutator variant (Pol+ Exo-), mutator variants with substitutions at Tyr567 in the polymerase active site (PolM Exo+), and the double mutator PolM Exo-. Comparing the mutational spectra of the Pol+ Exo- and Pol+ Exo+ enzymes revealed the patterns and efficiencies of proofreading, while Tyr567 was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.

Most colicins kill Escherichia coli cells by membrane pore formation or nuclease activity and, superficially, the mechanisms are similar: receptor binding, translocon recruitment, periplasmic receptor binding and membrane insertion. However, in detail, they employ a wide variety of molecular interactions that reveal a high degree of evolutionary diversification. Group A colicins bind to members of the TolQRAB complex in the periplasm and heterotrimeric complexes of colicin–TolA–TolB have been observed for both ColA and ColE9. ColN, the smallest and simplest pore-forming colicin, binds only to TolA and we show here that it uses the binding site normally used by TolB, effectively preventing formation of the larger complex used by other colicins. ColN binding to TolA was by β-strand addition with a KD of 1 µM compared with 40 µM for the TolA–TolB interaction. The β-strand addition and ColN activity could be abolished by single proline point mutations in TolA, which each removed one backbone hydrogen bond. By also blocking TolA–TolB binding these point mutations conferred a complete tol phenotype which destabilized the outer membrane, prevented both ColA and ColE9 activity, and abolished phage protein binding to TolA. These are the only point mutations known to have such pleiotropic effects and showed that the TolA–TolB β-strand addition is essential for Tol function. The formation of this simple binary ColN–TolA complex provided yet more evidence of a distinct translocation route for ColN and may help to explain the unique toxicity of its N-terminal domain.

The laboratory evolution of protease enzymes has the potential to generate proteases with therapeutically relevant specificities, and to assess the vulnerability of protease inhibitor drug candidates to the evolution of drug resistance. Here we describe a system for the continuous directed evolution of proteases using phage-assisted continuous evolution (PACE) that links the proteolysis of a target peptide to phage propagation through a protease-activated RNA polymerase (PA-RNAP). We use protease PACE in the presence of danoprevir or asunaprevir, two hepatitis C virus (HCV) protease inhibitor drug candidates in clinical trials, to continuously evolve HCV protease variants that exhibit up to 30-fold drug resistance in only 1 to 3 days of PACE. The predominant mutations evolved during PACE are mutations observed to arise in human patients treated with danoprevir or asunaprevir, demonstrating that protease PACE can rapidly identify the vulnerabilities of drug candidates to the evolution of clinically relevant drug resistance.

The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.

The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair. The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex. Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCl. In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5' to and three to five nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct. They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1. UvrABCI incises at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII. DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding. Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG-. These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage.

The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 degrees C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.

The response to hydrophobic mismatch of membrane-bound M13 major coat protein is measured using site-directed fluorescence and ESR spectroscopy. For this purpose, we investigate the membrane-anchoring interactions of M13 coat protein in model systems consisting of phosphatidylcholine bilayers that vary in hydrophobic thickness. Mutant coat proteins are prepared with an AEDANS-labeled single cysteine residue in the hinge region of the protein or at the C-terminal side of the transmembrane helix. In addition, the fluorescence of the tryptophan residue is studied as a monitor for the N-terminal side of the transmembrane helix. The fluorescence results show that the hinge region and C-terminal side of the transmembrane helix hardly respond to hydrophobic mismatch. In contrast, the N-terminal side of the helical transmembrane domain shifts to a more apolar environment, when the hydrophobic thickness is increased. The apparent strong membrane-anchoring interactions of the C-terminus are confirmed using a mutant that contains a longer transmembrane domain. As a result of this mutation, the tryptophan residue at the N-terminal side of the helical domain clearly shifts to a more polar environment, whereas the labeled position 46 at the C-terminal side is not affected. The phenylalanines in the C-terminal part of the protein play an important role in these apparent strong anchoring interactions. This is demonstrated with a mutant in which both phenylalanines are replaced by alanine residues. The phenylalanine residues in the C-terminus affect the location in the membrane of the entire transmembrane domain of the protein.

Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx2SLYP and Kx3NSxYG (TPR-1) and motifs Kx3NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of φ29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in φ29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer., This investigation was supported by Research Grant 2R01 GM27242-21 from the National Institutes of Health, by Grant n° PB98-0645 from Dirección General de Investigación Cientı́fica y Técnica, by Grant ERBFMRX CT97 0125 from European Union, and by an Institutional Grant from Fundación Ramón Areces. E. D. was holder of a post-doctoral fellowship from European Union (ERBFMRX CT97 0125), and M. de V. was a recipient of a post-doctoral fellowship from Comunidad Autónoma de Madrid.

Analyses of transmembrane domains of proteins have revealed that aromatic residues tend to cluster at or near the lipid-water interface of the membrane. To assess protein-membrane interactions of such residues, a viable mutant library was generated of the major coat protein of bacteriophage M13 (a model single membrane-spanning protein) in which one or the other of its interfacial tyrosine residues (Tyr-21 and Tyr-24) is mutated. Using the interfacial tryptophan (Trp-26) as an intrinsic probe, blue shifts in fluorescence emission spectra and quenching constants indicated that mutants with a polar amino acid substitution (such as Y24D or Y24N) are less buried in a deoxycholate micelle environment than in the wild type protein. These polar mutants also exhibited alpha-helix to beta-structure transition temperatures in incremental-heating circular dichroism studies relatively lower than those of wild type and nonpolar mutants (such as Y21V, Y21I, and Y24A), indicating that specific side chains in the lipid-water interface influence local protein-micelle interactions. Mutant Y21F exhibited the highest transition temperature, suggesting that phenylalanine is ostensibly the most effective interfacial anchoring residue. Using phage viability as the assay in a combination of site-directed and saturation mutagenesis experiments, it was further observed that both Tyr residues could not simultaneously be "knocked out". The overall results support the notion that an interfacial Tyr is a primary recognition element for precise strand positioning in vivo, a function that apparently cannot be performed optimally by residues with simple aliphatic character.

PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.