Structural plasticity and thermal stability of the histone-like protein from <i>Spiroplasma melliferum</i> are due to phenylalanine insertions into the conservative scaffold

<p>The histone-like (HU) protein is one of the major nucleoid-associated proteins of the bacterial nucleoid, which shares high sequence and structural similarity with IHF but differs from the latter in DNA-specificity. Here, we perform an analysis of structural-dynamic properties of HU protein from <i>Spiroplasma melliferum</i> and compare its behavior in solution to that of another mycoplasmal HU from <i>Mycoplasma gallisepticum</i>. The high-resolution heteronuclear NMR spectroscopy was coupled with molecular-dynamics study and comparative analysis of thermal denaturation of both mycoplasmal HU proteins. We suggest that stacking interactions in two aromatic clusters in the HUSpm dimeric interface determine not only high thermal stability of the protein, but also its structural plasticity experimentally observed as slow conformational exchange. One of these two centers of stacking interactions is highly conserved among the known HU and IHF proteins. Second aromatic core described recently in IHFs and IHF-like proteins is considered as a discriminating feature of IHFs. We performed an electromobility shift assay to confirm high affinities of HUSpm to both normal and distorted dsDNA, which are the characteristics of HU protein. MD simulations of HUSpm with alanine mutations of the residues forming the non-conserved aromatic cluster demonstrate its role in dimer stabilization, as both partial and complete distortion of the cluster enhances local flexibility of HUSpm.</p

Comparison of histone-like HU protein DNA-binding properties and HU/IHF protein sequence alignment

<div><p>Background</p><p>The structure and function of bacterial nucleoid are controlled by histone-like proteins of HU/IHF family, omnipresent in bacteria and also founding archaea and some eukaryotes.HU protein binds dsDNA without sequence specificity and avidly binds DNA structures with propensity to be inclined such as forks, three/four-way junctions, nicks, overhangs and DNA bulges. Sequence comparison of thousands of known histone-like proteins from diverse bacteria phyla reveals relation between HU/IHF sequence, DNA–binding properties and other protein features.</p><p>Methodology and principal findings</p><p>Performed alignment and clusterization of the protein sequences show that HU/IHF family proteins can be unambiguously divided into three groups, HU proteins, IHF_A and IHF_B proteins. HU proteins, IHF_A and IHF_B proteins are further partitioned into several clades for IHF and HU; such a subdivision is in good agreement with bacterial taxonomy. We also analyzed a hundred of 3D fold comparative models built for HU sequences from all revealed HU clades. It appears that HU fold remains similar in spite of the HU sequence variations. We studied DNA–binding properties of HU from <i>N</i>. <i>gonorrhoeae</i>, which sequence is similar to one of <i>E</i>.<i>coli</i> HU, and HU from <i>M</i>. <i>gallisepticum</i> and <i>S</i>. <i>melliferum</i> which sequences are distant from <i>E</i>.<i>coli</i> protein. We found that in respect to dsDNA binding, only <i>S</i>. <i>melliferum</i> HU essentially differs from <i>E</i>.<i>coli</i> HU. In respect to binding of distorted DNA structures, <i>S</i>. <i>melliferum</i> HU and <i>E</i>.<i>coli</i> HU have similar properties but essentially different from <i>M</i>. <i>gallisepticum</i> HU and <i>N</i>. <i>gonorrhea</i> HU. We found that in respect to dsDNA binding, only <i>S</i>. <i>melliferum</i> HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than <i>E</i>.<i>coli</i> and <i>N</i>. <i>gonorrhoeae</i>. In respect to binding to distorted DNA structures, each HU protein has its individual profile of affinities to various DNA-structures with the increased specificity to DNA junction.</p><p>Conclusions and significance</p><p>HU/IHF family proteins sequence alignment and classification are updated. Comparative modeling demonstrates that HU protein 3D folding’s even more conservative than HU sequence. For the first time, DNA binding characteristics of HU from <i>N</i>. <i>gonorrhoeae</i>, <i>M</i>. <i>gallisepticum</i> and <i>S</i>. <i>melliferum</i> are studied. Here we provide detailed analysis of the similarity and variability of DNA-recognizing and bending of four HU proteins from closely and distantly related HU clades.</p></div

Model of <i>E</i>.<i>coli</i> HUα dimer with hotspots for amino acid insertions and deletions.

<p>Each HU monomer contains three alpha helixes and five beta strands. HU body (helixes 1 and 2) is responsible for dimer stabilization. HU arms are responsible for DNA binding. Hotspots for amino acid insertions and deletions in HU are shown.</p

Model of HU <i>Bifidobacterium longum</i> from Actinobacteria (clade HU_acti_0), superimposed with <i>E</i>. <i>coli</i> HUα model.

<p><i>B</i>. <i>longum</i> HU is shown in yellow, <i>E</i>. <i>coli</i> HUα—in cyan. Angle between alpha helix 2 and alpha helix 3 is shown in magenta.</p

Model of <i>Pseudomonas syringae</i> HU (magenta) superimposed with <i>E</i>. <i>coli</i> HUα model (cyan).

<p>Angle between alpha helix 1 and alpha helix 2 is shown in green.</p

HU binding to dsDNA of various lengths.

<p>Binding of labeled DNA to HU proteins was analyzed by polyacrylamide gel electrophoresis. The gel was buffered with 50 mM Tris–borate; binding mixture contains 40 mM NaCl. DNA samples were: dsDNA of sequence ‘D’ with the length varying from 21 to 48 bp (indicated at the bottom). HU origin and concentration is indicated at the top (“-“, no HU was added). Bands corresponding to HU-DNA complexes are marked with arrows, the number of HU dimers in each complex is indicated on the left of the arrow. Panels correspond to HU proteins of various bacteria, protein concentrations are indicated.</p

HU binding to “distorted” DNA structures checked by polyacrylamide gel mobility assay.

<p>HU protein at concentrations indicated above the gel image (“-“, no HU was added) was mixed with 5’-labelled DNA in a buffer containing 150 mM NaCl; the bound and free DNA were gel-separated. DNA structures indicated at the bottom of the gel images: n, nicked DNA; ds, dsDNA; A1, A3 and A7, DNA bulges, containing one, three or seven non-paired adenines in one of DNA strands; J–four-way junction; fork, ssDNA fork; ov, DNA overhang; iJ, incomplete junction lacking one DNA strand; inv, DNA invasion. Panels correspond to HU proteins of various bacteria.</p

Consensus sequences of three IHF_A, three IHF_B, and several HU clades revealed in this study.

<p>Conservative residues are highlighted. Alpha helixes and beta sheets are indicated with yellow and blue blocks, respectively.</p

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 107 independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms