Potent and broad HIV-1 neutralization in fusion peptide-primed SHIV-infected macaques.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID] ∼100) on a 208-strain panel.

Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site.

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display.

Development of bright red-shifted miRFP704nano using structural analysis of miRFPnano proteins.

We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.

Vaccine elicitation and structural basis for antibody protection against alphaviruses.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups.

Crystal Structure of Bright Fluorescent Protein BrUSLEE with Subnanosecond Fluorescence Lifetime; Electric and Dynamic Properties.

The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component of fluorescence lifetime (FL~0.

Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein.

Applying rational design, we developed 17 kDa cyanobacteriochrome-based near-infrared (NIR-I) fluorescent protein, miRFP718nano. miRFP718nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues. miRFP718nano has maximal emission at 718 nm and an emission tail in the short-wave infrared (SWIR) region, allowing deep-penetrating off-peak fluorescence imaging in vivo.

Single-domain near-infrared protein provides a scaffold for antigen-dependent fluorescent nanobodies.

Small near-infrared (NIR) fluorescent proteins (FPs) are much needed as protein tags for imaging applications. We developed a 17 kDa NIR FP, called miRFP670nano3, which brightly fluoresces in mammalian cells and enables deep-brain imaging. By exploring miRFP670nano3 as an internal tag, we engineered 32 kDa NIR fluorescent nanobodies, termed NIR-Fbs, whose stability and fluorescence strongly depend on the presence of specific intracellular antigens.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I.

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design.

For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein.

Structure-Based Rational Design of Two Enhanced Bacterial Lipocalin Tags for Protein-PAINT Super-resolution Microscopy.

Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes.

Bacterial Phytochrome as a Scaffold for Engineering of Receptor Tyrosine Kinases Controlled with Near-Infrared Light.

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm.

Two independent routes of post-translational chemistry in fluorescent protein FusionRed.

The crystal structure of monomeric red fluorescent protein FusionRed (λ/λ 580/608 mn) has been determined at 1.09 Å resolution and revealed two alternative routes of post-translational chemistry, resulting in distinctly different products. The refinement occupancies suggest the 60:40 ratio of the mature Met63-Tyr64-Gly65 chromophore and uncyclized chromophore-forming tripeptide with the protein backbone cleaved between Met63 and the preceding Phe62 and oxidized Cα-Cβ bond of Tyr64.

Crystallographic and calorimetric analysis on Pleurotus ostreatus lectin and its sugar complexes - promiscuous binding driven by geometry.

Carbohydrate recognition is established as a property of lectins and implicated in many functions including immunity and defense against pathogens. Many lectins are characterized and proposed for various applications owing to the above said recognition. The crystal structure of a lectin from Pleurotus ostreatus has been determined and shown to be calcium dependent.

Structural Factors Enabling Successful GFP-Like Proteins with Alanine as the Third Chromophore-Forming Residue.

GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine, and efforts to replace it were in vain.

Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing.

From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.

Crystal structure of a novel Kunitz type inhibitor, alocasin with anti-Aedes aegypti activity targeting midgut proteases.

Background: The pesticidal properties of many Kunitz-type inhibitors have been reported previously; however, the mechanism of action is not well established. In this study, the activity of alocasin against Aedes aegypti is demonstrated and the structure-activity relationship of this Kunitz-type inhibitor is explained through X-ray structure analyses.

Results: Alocasin was purified from mature rhizomes of Alocasia as a single polypeptide chain of ∼ 20 kDa.

Structural insights on starch hydrolysis by plant β-amylase and its evolutionary relationship with bacterial enzymes.

The conversion of starch to maltose is catalysed in plants by β-amylase. The enzymatic mechanism has been well-characterized for the soybean and barley enzymes, which utilise a glutamic acid-glutamate pair. In the present study, we present a surprise observation of maltotetraose at the active site, the presence of which elucidates the clear role of Thr344 as a conformational "switch" between substrate binding and product release during hydrolysis.

Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies.

Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties.

Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms.

The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm).

Structural analysis of β-prism lectin from Colocasia esculenta (L.) S chott.

The Mannose-binding β-Prism Colocasia esculenta lectin (β-PCL) was purified from tubers using ion exchange chromatography. The purified β-PCL appeared as a single band of ∼12kDa on SDS-PAGE. β-PCL crystallizes in trigonal space group P3121 and diffracted to a resolution of 2.

Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore.

Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.

Molecular Basis of Spectral Diversity in Near-Infrared Phytochrome-Based Fluorescent Proteins.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are the probes of choice for deep-tissue imaging. Detection of several processes requires spectrally distinct NIR FPs. We developed an NIR FP, BphP1-FP, which has the most blue-shifted spectra and the highest fluorescence quantum yield among BphP-derived FPs.

Structure of the green fluorescent protein NowGFP with an anionic tryptophan-based chromophore.

A green-emitting fluorescent variant, NowGFP, with a tryptophan-based chromophore (Thr65-Trp66-Gly67) was recently developed from the cyan mCerulean by introducing 18 point mutations. NowGFP is characterized by bright green fluorescence at physiological and higher pH and by weak cyan fluorescence at low pH. Illumination with blue light induces irreversible photoconversion of NowGFP from a green-emitting to a cyan-emitting form.

[Three dimensional structure of the dimeric gene-engineered variant of green fluorescent protein EGFP-K162Q in P6(1) crystal space group].

The crystal structure of the dimeric green fluorescent protein EGFP-K162Q with C-terminal deletion MDELYK (EGFPv) has been determined in space group P6 at resolution 1.34 A. The obtained structure has been compared with that of the monomeric form of EGFP (green biomarker with enhanced photophysical properties) determined in other crystal space group P2(1)2(1)2(1) at resolution 1.