Biomedical subjects

Karen L Maxwell

Publications and source records attributed to Karen L Maxwell.

8 recordsLinked to original sources

PubMed2006

The NMR structure of the gpU tail-terminator protein from bacteriophage lambda: identification of sites contributing to Mg(II)-mediated oligomerization and biological function.

During the late stages of lambda bacteriophage assembly, the protein gpU terminates tail polymerization and participates at the interface between the mature capsid and tail components. When it engages the lambda tail, gpU undergoes a monomer-hexamer transition to achieve its biologically active form. Towards understanding how gpU participates in multiple protein-protein interactions, we have solved the structure of gpU in its monomeric state using NMR methods. The structure reveals a mixed alpha/beta motif with several dynamic loops at the periphery. Addition of 20 mM MgCl(2) is known to oligomerize gpU in the absence of its protein partners. Multiple image analysis of electron micrographs revealed ring-like structures of magnesium ion saturated gpU with a 30 A pore, consistent with its function as a portal for the passage of viral DNA into the host bacterium. The ability of magnesium ions to promote oligomerization was lost when substitutions were made at a cluster of acidic amino acids in the vicinity of helix alpha2 and the beta1-beta2 loop. Furthermore, substitutions at these sites abolished the biological activity of gpU.

Amino Acid Sequence↗

PubMed2006

Ig-like domains on bacteriophages: a tale of promiscuity and deceit.

The immunoglobulin (Ig) fold is one of the most important structures in biology, playing essential roles in the vertebrate immune response, cell adhesion, and many other processes. Through bioinformatic analysis, we have discovered that Ig-like domains are often found in the constituent proteins of tailed double-stranded (ds) DNA bacteriophage particles, and are likely displayed on the surface of these viruses. These phage Ig-like domains fall into three distinct sequence families, which are similar to the classic immunoglobulin domain (I-Set), the fibronectin type 3 repeat (FN3), and the bacterial Ig-like domain (Big2). The phage Ig-like domains are very promiscuous. They are attached to more than ten different functional classes of proteins, and found in all three morphogenetic classes of tailed dsDNA phages. In addition, they reside in phages that infect a diverse set of gram negative and gram positive bacteria. These domains are deceptive because many are added to larger proteins through programmed ribosomal frameshifting, so that they are not always detected by standard protein sequence searching procedures. In addition, the presence of unrecognized Ig-like domains in a variety of phage proteins with different functions has led to gene misannotation. Our results demonstrate that horizontal gene transfer involving Ig-like domain encoding DNA has occurred commonly between diverse classes of both lytic and temperate phages, which otherwise display very limited sequence similarities to one another. We suggest that phage may have been an important vector in the spread of Ig-like domains through diverse species of bacteria. While the function of the phage Ig-like domains is unknown, several lines of evidence suggest that they may play an accessory role in phage infection by weakly interacting with carbohydrates on the bacterial cell surface.

Amino Acid Sequence↗

PubMed2005

Functional similarities between phage lambda Orf and Escherichia coli RecFOR in initiation of genetic exchange.

Genetic recombination in bacteriophage lambda relies on DNA end processing by Exo to expose 3'-tailed strands for annealing and exchange by beta protein. Phage lambda encodes an additional recombinase, Orf, which participates in the early stages of recombination by supplying a function equivalent to the Escherichia coli RecFOR complex. These host enzymes assist loading of the RecA strand exchange protein onto ssDNA coated with ssDNA-binding protein. In this study, we purified the Orf protein, analyzed its biochemical properties, and determined its crystal structure at 2.5 angstroms. The homodimeric Orf protein is arranged as a toroid with a shallow U-shaped cleft, lined with basic residues, running perpendicular to the central cavity. Orf binds DNA, favoring single-stranded over duplex and with no obvious preference for gapped, 3'-tailed, or 5'-tailed substrates. An interaction between Orf and ssDNA-binding protein was indicated by far Western analysis. The functional similarities between Orf and RecFOR are discussed in relation to the early steps of recombinational exchange and the interplay between phage and bacterial recombinases.

Amino Acid Sequence↗

PubMed2005

Crystal structure of bacteriophage lambda cII and its DNA complex.

The tetrameric cII protein from bacteriophage lambda activates transcription from the phage promoters P(RE), P(I), and P(AQ) by binding to two direct repeats that flank the promoter -35 element. Here, we present the X-ray crystal structure of cII alone (2.8 A resolution) and in complex with its DNA operator from P(RE) (1.7 A resolution). The structures provide a basis for modeling of the activation complex with the RNA polymerase holoenzyme, and point to the key role for the RNA polymerase alpha subunit C-terminal domain (alphaCTD) in cII-dependent activation, which forms a bridge of protein/protein interactions between cII and the RNA polymerase sigma subunit. The model makes specific predictions for protein/protein interactions between cII and alphaCTD, and between alphaCTD and sigma, which are supported by previous genetic studies.

Bacteriophage lambda↗

PubMed2005

Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins.

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

Biochemistry↗

PubMed2003

Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins.

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (<or=18 kD) that are normally soluble in vivo. In addition, refolding the purified proteins by using dialysis against a single buffer allowed us to obtain soluble protein samples of 58% of small proteins that were found in the insoluble fraction in vivo, and 10% of the initial number of proteins provided good heteronuclear single quantum coherence (HSQC) NMR spectra. We conclude that a denaturation/refolding protocol is an efficient way to generate structural samples for high-throughput studies of small proteins.

Biophysical Phenomena↗

PubMed2002

Protein folding kinetics beyond the phi value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state.

The SH3 domain folding transition state structure contains two well-ordered turn regions, known as the diverging turn and the distal loop. In the Src SH3 domain transition state, these regions are stabilized by a hydrogen bond between Glu30 in the diverging turn and Ser47 in the distal loop. We have examined the effects on folding kinetics of amino acid substitutions at the homologous positions (Glu24 and Ser41) in the Fyn SH3 domain. In contrast to most other folding kinetics studies which have focused primarily on non-disruptive substitutions with Ala or Gly, here we have examined the effects of substitutions with diverse amino acid residues. Using this approach, we demonstrate that the transition state structure is generally tolerant to amino acid substitutions. We also uncover a unique role for Ser at position 41 in facilitating folding of the distal loop, which can only be replicated by Asp at the same position. Both these residues appear to accelerate folding through the formation of short-range side-chain to backbone hydrogen bonds. The folding of the diverging turn region is shown to be driven primarily by local interactions. The diverging turn and distal loop regions are found to interact in the transition state structure, but only in the context of particular mutant backgrounds. This work demonstrates that studying the effects of a variety of amino acid substitutions on protein folding kinetics can provide unique insights into folding mechanisms which cannot be obtained by standard Phi value analysis.

Amino Acids↗

PubMed2002

The solution structure of the bacteriophage lambda head-tail joining protein, gpFII.

The bacteriophage lambda FII protein (gpFII) is a 117 residue structural protein found in the phage particle that is required for the joining of phage heads and tails at the last step of morphogenesis. We have performed biophysical experiments to show that gpFII is stable, monomeric, and reversibly folded. We have also determined the atomic resolution structure of gpFII using NMR spectroscopy. gpFII is shown to possess a novel fold consisting of seven beta-strands and a short alpha-helix. It also displays two large unstructured regions at the N terminus (residues 1-24) and in a large loop near the middle of the protein (residues 46-62). We speculate that these unstructured regions become structured when gpFII assembles into the phage particle, and that these conformational changes play an important role in regulating the assembly pathway. Alignment of the gpFII sequence with those of homologues from other lambdoid phages has allowed us to putatively identify distinct surfaces on the gpFII structure that mediate binding to the phage head and tail.

Amino Acid Sequence↗