Phage display technology provides a versatile tool for exploring the interactions between proteins, peptides and small molecule ligands. Quantitative analysis of peptide population sequence diversity ...and bias patterns has the power to significantly enhance the impact of these methods 1, 2. We have developed a suite of computational tools for the analysis of peptide populations and made them accessible by integrating fifteen software programs for the analysis of combinatorial peptide sequences into the REceptor LIgand Contacts (RELIC) relational database and web‐server. These programs have been developed for the analysis of statistical properties of peptide populations; identification of weak consensus sequences within these populations; and the comparison of these peptide sequences to those of naturally occurring proteins. RELIC is particularly suited to the analysis of peptide populations affinity selected with a small molecule ligand such as a drug or metabolite. Within this functional context, the ability to identify potential small molecule binding proteins using combinatorial peptide screening will accelerate as more ligands are screened and more genome sequences become available. The broader impact of this work is the addition of a novel means of analyzing peptide populations to the phage display community.
We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and ...filamentous phage morphogenesis. Using a bioinformatics‐assisted computational approach, collections of random peptide sequences obtained from a T7 12‐mer library (X12) and a T7 7‐mer disulfide‐constrained library (CX7C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.‐12™ and Ph.D.‐C7C™ libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7‐displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7‐displayed peptide libraries were screened for streptavidin‐ and neutravidin‐binding phage. Novel binding motifs were identified for each protein.
X-ray solution scattering in both the small-angle (SAXS) and wide-angle (WAXS) regimes is making an increasing impact on our understanding of biomolecular complexes. The accurate calculation of WAXS ...patterns from atomic coordinates has positioned the approach for rapid growth and integration with existing Structural Genomics efforts. WAXS data are sensitive to small structural changes in proteins; useful for calculation of the pair-distribution function at relatively high resolution; provides a means to characterize the breadth of the structural ensemble in solution; and can be used to identify proteins with similar folds. WAXS data are often used to test structural models, identify structural similarities and characterize structural changes. WAXS is highly complementary to crystallography and NMR. It holds great potential for the testing of structural models of proteins; identification of proteins that may exhibit novel folds; characterization of unfolded or natively disordered proteins; and detection of structural changes associated with protein function.
An alternate formulation of helical diffraction theory is used to generate cross-sectional shapes of fibrous structures from equatorial scattering. We demonstrate this approach with computationally ...generated scattering intensities and then apply it to scattering data from Tobacco Mosaic Virus (TMV) and in vitro assembled fibrils of Aβ40 peptides. Refining the cross-sectional shape of TMV from SAXS data collected on a 26mg/ml solution resulted in a circular shape with outer diameter of ∼180Å and inner diameter of ∼40Å consistent with the known structure of TMV. We also utilized this method to analyze the equatorial scattering from TMV collected by Don Caspar from a concentrated (24% ∼295mg/ml) gel of TMV as reported in his Ph.D. thesis in 1955. This data differs from the SAXS data in having a sharp interference peak at ∼250Å spacing, indicative of strong interparticle interactions in the gel. Analysis of this data required consideration of interatomic vectors as long as 2000Å and resulted in generation of images that were interpreted as representative of local organization of TMV particles in the sample. Peaks in the images were separated, on average by about 250Å with a density consistent with Caspar’s original measurements. Analysis of SAXS data from Aβ fibrils resulted in a cross-sectional shape that could be interpreted in terms of structural models that have been constructed from ssNMR and cryoEM. These results demonstrate an unexpected use of the small-angle region of fiber diffraction patterns to derive fundamental structural properties of scattering objects.
In the field of small‐angle X‐ray scattering (SAXS), the task of estimating the size of particles in solution is usually synonymous with the Guinier plot. The approximation behind this plot, ...developed by Guinier in 1939, provides a simple yet accurate characterization of the scattering behavior of particles at low scattering angle or momentum transfer q, together with a computationally efficient way of inferring their radii of gyration RG. Moreover, this approximation is valid beyond spherical scatterers, making its use ubiquitous in the SAXS world. However, when it is important to estimate further particle characteristics, such as the anisotropy of the scatterer's shape, no similar or extended approximations are available. Existing tools to characterize the shape of scatterers rely either on prior knowledge of the scatterers' geometry or on iterative procedures to infer the particle shape ab initio. In this work, a low‐angle approximation of the scattering intensity I(q) for ellipsoids of revolution is developed and it is shown how the size and anisotropy information can be extracted from the parameters of that approximation. The goal of the approximation is not to estimate a particle's full structure in detail, and thus this approach will be less accurate than well known iterative and ab initio reconstruction tools available in the literature. However, it can be considered as an extension of the Guinier approximation and used to generate initial estimates for the aforementioned iterative techniques, which usually rely on RG and Dmax for initialization. This formulation also demonstrates that nonlinearity in the Guinier plot can arise from anisotropy in the scattering particles. Beyond ideal ellipsoids of revolution, it is shown that this approximation can be used to estimate the size and shape of molecules in solution, in both computational and experimental scenarios. The limits of the approach are discussed and the impact of a particle's anisotropy in the Guinier estimate of RG is assessed.
In this work, a Guinier‐like approximation to directly estimate the anisotropy of molecules in solution from their small‐angle X‐ray scattering intensity around the origin is developed. This approach provides deeper insight into a molecule's structure than the traditional Guinier RG estimation and can be used in ab initio reconstruction methods to provide better initialization strategies.
Bacterial adhesion pili are designed to bind specifically and maintain attachment of bacteria to target cells. Uropathogenic P-pili are sufficiently mechanically resilient to resist the cleansing ...action of urine flow that removes most other bacteria. P-pili are 68 A in diameter and approximately 1 micron long, and are composed of approximately 1,000 copies of the principal structural protein, PapA. They are attached to the outer membrane by a minor structural protein, PapH and are terminated by an approximately 20 A diameter fibrillus composed of PapK, PapE and PapF, which presents the host-binding adhesin PapG. The amino-acid sequences of PapA, PapE, and PapF are similar, with highly conserved C-termini being responsible for binding to PapD, the periplasmic chaperone. Our three-dimensional reconstruction indicates that pili are formed by the tight winding of a much thinner structure. A structural transition allows the pilus to unravel without depolymerizing, producing a thin, extended structure five times the length of the original pilus.
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