The polaroid image as photo-object

This article is part of a larger project on the cultural history of Polaroid photography and draws on research done at the Polaroid Corporate archive at Harvard and at the Polaroid company itself. It identifies two cultural practices engendered by Polaroid photography, which, at the point of its extinction, has briefly flared into visibility again. It argues that these practices are mistaken as novel but are in fact rediscoveries of practices that stretch back as many as five decades. The first section identifies Polaroid image-making as a photographic equivalent of what Tom Gunning calls the ‘cinema of attractions’. That is, the emphasis in its use is on the display of photographic technologies rather than the resultant image. Equally, the common practice, in both fine art and vernacular circles, of making composite pictures with Polaroid prints, draws attention from image content and redirects it to the photo as object

A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ∼10% or less of wild-type, while the other has ∼45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues

Structure and Dynamics of the G121V Dihydrofolate Reductase Mutant: Lessons from a Transition-State Inhibitor Complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on 15N and 2H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect

Probing the Flexibility of Large Conformational Changes in Protein Structures through Local Perturbations

Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate—the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of α-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen Receptor ligand-binding domain, the RIP deformation map is quite sparse except for one large conformational change involving Helix-12, which is the structural element that allosterically links ligand binding to receptor activation. RIP analysis has the potential to discover sites of functional conformational changes and the linchpin residues critical in determining these conformational states

Specific rotation of yeast adenylic acid in anhydrous formamide /

Work performed at Washington University."AECU-581"Includes bibliographic references.Mode of access: Internet

Glutathione-S-transferase FosA6 of klebsiella pneumoniae origin conferring fosfomycin resistance in ESBL-producing Escherichia coli

Objectives: The objectives of this study were to elucidate the genetic context of a novel plasmid-mediated fosA variant, fosA6, conferring fosfomycin resistance and to characterize the kinetic properties of FosA6.Methods: The genome of fosfomycin-resistant Escherichia coli strain YD786 was sequenced. Homologues of FosA6 were identified through BLAST searches. FosA6 and FosAST258 were purified and characterized using a steady-state kinetic approach. Inhibition of FosA activity was examined with sodium phosphonoformate.Results: Plasmid-encoded glutathione-S-transferase (GST) FosA6 conferring high-level fosfomycin resistance was identified in a CTX-M-2-producing E. coli clinical strain at a US hospital. fosA6 was carried on a self-conjugative, 69 kb IncFII plasmid. The ΔlysR-fosA6-ΔyjiR_1 fragment, located between IS10R and ΔIS26, was nearly identical to those on the chromosomes of some Klebsiella pneumoniae strains (MGH78578, PMK1 and KPPR1). FosA6 shared >99% identity with chromosomally encoded FosAPMK1 in K. pneumoniae of various STs and 98% identity with FosAST258, which is commonly found in K. pneumoniae clonal complex (CC) 258 including ST258. FosA6 and FosAST258 demonstrated robust GST activities that were comparable to each other. Sodium phosphonoformate, a GST inhibitor, reduced the fosfomycin MICs by 6- to 24-fold for K. pneumoniae and E. coli strains carrying fosA genes on the chromosomes and plasmids, respectively.Conclusions: FosA6, probably captured from the chromosome of K. pneumoniae, conferred high-level fosfomycin resistance in E. coli. FosA6 functioned as a GST and inactivated fosfomycin efficiently. K. pneumoniae may serve as a reservoir of fosfomycin resistance for E. coli

Genomic classification and antimicrobial resistance profiling of Streptococcus pneumoniae and Haemophilus influenzae isolates associated with paediatric otitis media and upper respiratory infection

Abstract Acute otitis media (AOM) is the most common childhood bacterial infectious disease requiring antimicrobial therapy. Most cases of AOM are caused by translocation of Streptococcus pneumoniae or Haemophilus influenzae from the nasopharynx to the middle ear during an upper respiratory tract infection (URI). Ongoing genomic surveillance of these pathogens is important for vaccine design and tracking of emerging variants, as well as for monitoring patterns of antibiotic resistance to inform treatment strategies and stewardship. In this work, we examined the ability of a genomics-based workflow to determine microbiological and clinically relevant information from cultured bacterial isolates obtained from patients with AOM or an URI. We performed whole genome sequencing (WGS) and analysis of 148 bacterial isolates cultured from the nasopharynx (N = 124, 94 AOM and 30 URI) and ear (N = 24, all AOM) of 101 children aged 6–35 months presenting with AOM or an URI. We then performed WGS-based sequence typing and antimicrobial resistance profiling of each strain and compared results to those obtained from traditional microbiological phenotyping. WGS of clinical isolates resulted in 71 S. pneumoniae genomes and 76 H. influenzae genomes. Multilocus sequencing typing (MSLT) identified 33 sequence types for S. pneumoniae and 19 predicted serotypes including the most frequent serotypes 35B and 3. Genome analysis predicted 30% of S. pneumoniae isolates to have complete or intermediate penicillin resistance. AMR predictions for S. pneumoniae isolates had strong agreement with clinical susceptibility testing results for beta-lactam and non beta-lactam antibiotics, with a mean sensitivity of 93% (86–100%) and a mean specificity of 98% (94–100%). MLST identified 29 H. influenzae sequence types. Genome analysis identified beta-lactamase genes in 30% of H. influenzae strains, which was 100% in agreement with clinical beta-lactamase testing. We also identified a divergent highly antibiotic-resistant strain of S. pneumoniae, and found its closest sequenced strains, also isolated from nasopharyngeal samples from over 15 years ago. Ultimately, our work provides the groundwork for clinical WGS-based workflows to aid in detection and analysis of H. influenzae and S. pneumoniae isolates

Structural insights into the molecular mechanism of high-level ceftazidime–avibactam resistance conferred by CMY-185

ABSTRACTβ-Lactamases can accumulate stepwise mutations that increase their resistance profiles to the latest β-lactam agents. CMY-185 is a CMY-2-like β-lactamase and was identified in an Escherichia coli clinical strain isolated from a patient who underwent treatment with ceftazidime-avibactam. CMY-185, possessing four amino acid substitutions of A114E, Q120K, V211S, and N346Y relative to CMY-2, confers high-level ceftazidime-avibactam resistance, and accumulation of the substitutions incrementally enhances the level of resistance to this agent. However, the functional role of each substitution and their interplay in enabling ceftazidime-avibactam resistance remains unknown. Through biochemical and structural analysis, we present the molecular basis for the enhanced ceftazidime hydrolysis and impaired avibactam inhibition conferred by CMY-185. The substituted Y346 residue is a major driver of the functional evolution as it rejects primary avibactam binding due to the steric hindrance and augments oxyimino-cephalosporin hydrolysis through a drastic structural change, rotating the side chain of Y346 and then disrupting the H-10 helix structure. The other substituted residues E114 and K120 incrementally contribute to rejection of avibactam inhibition, while S211 stimulates the turnover rate of the oxyimino-cephalosporin hydrolysis. These findings indicate that the N346Y substitution is capable of simultaneously expanding the spectrum of activity against some of the latest β-lactam agents with altered bulky side chains and rejecting the binding of β-lactamase inhibitors. However, substitution of additional residues may be required for CMY enzymes to achieve enhanced affinity or turnover rate of the β-lactam agents leading to clinically relevant levels of resistance.IMPORTANCECeftazidime-avibactam has a broad spectrum of activity against multidrug-resistant Gram-negative bacteria including carbapenem-resistant Enterobacterales including strains with or without production of serine carbapenemases. After its launch, emergence of ceftazidime-avibactam-resistant strains that produce mutated β-lactamases capable of efficiently hydrolyzing ceftazidime or impairing avibactam inhibition are increasingly reported. Furthermore, cross-resistance towards cefiderocol, the latest cephalosporin in clinical use, has been observed in some instances. Here, we clearly demonstrate the functional role of the substituted residues in CMY-185, a four amino-acid variant of CMY-2 identified in a patient treated with ceftazidime-avibactam, for high-level resistance to this agent and low-level resistance to cefiderocol. These findings provide structural insights into how β-lactamases may incrementally alter their structures to escape multiple advanced β-lactam agents