Protein minimization by random fragmentation and selection

Rudgers, Gary W.; Palzkill, Timothy

doi:10.1093/protein/14.7.487

Cited by 17 publications

(10 citation statements)

References 33 publications

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“…Additionally, the convergence of clones retained by the rAtPIMT1 in the presence of AdoMet to a few identical species during successive rounds of biopanning (Fig. 3b) is a hallmark of success in this endeavor (52), suggesting that these clones were particularly susceptible to isoAsp formation, out-competing others for available rAtPIMT1. This fact also necessitated re-commencement of the biopan with the naïve libraries after a (several) target had been recovered in the fourth biopanning round.…”

Section: Discussionmentioning

confidence: 99%

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

Chen

Nayak

Majee

et al. 2010

Journal of Biological Chemistry

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The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by onblot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An overrepresentation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

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Section: Discussionmentioning

confidence: 99%

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

Chen

Nayak

Majee

et al. 2010

Journal of Biological Chemistry

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“…Peptides corresponding to residues 46 -51 of BLIP (the Asp-49 binding loop) were shown to bind TEM-1 and SHV-1 with affinities of 488 and 420 M, respectively (37). Random fragmentation and phage display of BLIP identified a peptide consisting of residues 30 -49 of BLIP that inhibits TEM-1 with 446 M affinity, and a combination of phage display and peptide arrays identified a 136 M inhibitor that has 50% sequence identity to BLIP residues 46 -51 (38,39). All of these studies point to the central importance of the Asp-49 loop in inhibition.…”

Section: Discussionmentioning

confidence: 99%

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Reynolds

Thomson

Corbett

et al. 2006

Journal of Biological Chemistry

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␤-Lactamase inhibitor protein (BLIP) binds a variety of class A ␤-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 ␤-lactamases are almost structurally identical, BLIP binds TEM-1 ϳ1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of ␤-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6 Å resolution crystal structure of SHV-1⅐BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV⅐BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1⅐BLIP structure with the published TEM-1⅐BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8 Å SHV D104K⅐BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant ␤-lactamase⅐BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/ mol of the experimental values for 112 mutations at the TEM-1⅐BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1⅐BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the ␤-lactamase⅐BLIP complexes and computationally assisted design of tight binding BLIP variants.Class A ␤-lactamases are a major cause of ␤-lactam resistance in Gram-negative bacteria. These enzymes catalyze the hydrolysis of ␤-lactam antibiotics, such as penicillins and cephalosporins, rendering them inactive. ␤-Lactamase inhibitor protein (BLIP), 6 which is secreted by the Gram-positive soil bacterium Streptomyces clavuligeris, inhibits a variety of class A ␤-lactamase enzymes with a wide spectrum of affinities. Its binding partners include Escherichia coli TEM-1, Klebsiella pneumoniae SHV-1, Serratia marcescens SME-1, Bacillus anthracis BlaI, and Proteus vulgaris K1, among others. BLIP is able to inhibit K1 with picomolar affinity and TEM-1, SME-1, and BlaI with nanomolar affinity. However, it inhibits SHV-1 with only micromolar affinity (1, 2). Whereas SHV-1 shares 67% sequence identity with TEM-1 (Fig. 1), and the crystal structures of the unbound ␤-lactamases overlay with an ␣-carbon r.m.s. deviation of 1.4 Å, BLIP exhibits a 1000-fold difference in affinity for the two (3). This poses an interesting question of binding specificity and affinity. How does BLIP bind multiple targets, and what is the source of variation in binding affinity? Recent alanine scanning mutagenesis has provided insight into the origins of BLIP affinity and specificity for an array of ␤-lactamases, including TEM-1 and SHV-1 (2, 4). However, interpretation of these data has been limited, because only the structure of the T...

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“…15,[17][18][19]21,24,25 In addition, several inhibitory peptides have been designed from key interacting residues of BLIP. [26][27][28][29] It has been consistently noted in the literature that the biological function of BLIP is to inhibit β-lactamases in an escalating microbiological arms race. Yet this has not been proven.…”

Section: Introductionmentioning

confidence: 99%

Insights into Positive and Negative Requirements for Protein–Protein Interactions by Crystallographic Analysis of the β-Lactamase Inhibitory Proteins BLIP, BLIP-I, and BLP

Gretes

Lim

Castro

et al. 2009

Journal of Molecular Biology

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Protein minimization by random fragmentation and selection

Cited by 17 publications

References 33 publications

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Insights into Positive and Negative Requirements for Protein–Protein Interactions by Crystallographic Analysis of the β-Lactamase Inhibitory Proteins BLIP, BLIP-I, and BLP

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