scholarly journals REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants

2019 ◽  
Author(s):  
Kevin Dalton ◽  
Tom Lopez ◽  
Vijay Pande ◽  
Judith Frydman
Keyword(s):  
Rational Design ◽  
Biochemical Characterization ◽  
Cysteine Residue ◽  
Rational Approach ◽  
Protein Activity ◽  
Methanococcus Maripaludis ◽  
Site Specific ◽  
Protein Variants ◽  
Group Ii Chaperonin ◽  
Reduced Activity

AbstractSite-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.

Site-specific Labeling of Active Proteins with Gold Nanoparticles

2005 ◽  
Vol 900 ◽  
Author(s):  
Marie-Eve Aubin-Tam ◽  
Kimberly Hamad-Schifferli
Keyword(s):  
Cytochrome C ◽  
Gold Nanoparticle ◽  
Cysteine Residue ◽  
Specific Adsorption ◽  
Covalent Attachment ◽  
Protein Activity ◽  
Visible Absorption ◽  
Site Specific ◽  
Reaction Stoichiometry ◽  
Positively Charged

ABSTRACTCovalent conjugation of nanoparticles to proteins is challenging as proteins have numerous residues to which the nanoparticle can non-specifically adsorb. This is problematic as non-specific adsorption is known to denature the protein, altering its structure and thus compromising protein activity. We study site specific gold nanoparticle labeling of two enzymes, Ribonuclease S and Cytochrome c, with the goal of understanding conditions that minimize non-specific adsorption and optimize protein structure and activity. Ribonuclease S is a two-piece protein made of S-peptide and S-protein. 3nm gold nanoparticle is attached to a mutated cysteine residue on the S-peptide. The altered enzymatic activity of gold labeled Ribonuclease S is determined using RNA substrate with a fluorophore-quencher couple. Cytochrome c is linked to 1.5nm nanoparticles with ligands having neutral, negatively, or positively charged endgroups, through covalent attachment of gold with a specific surface cysteine residue. The labeled protein is characterized by circular dichroism spectroscopy and UV-visible absorption. For both proteins, agarose gel electrophoresis was used to determine optimal reaction stoichiometry and also probe non-specific adsorption between the nanoparticle and protein.

scholarly journals Rational Design of Self-assembling Artificial Proteins Utilizing a Micelle-Assisted Protein Labeling Technology (MAPLabTech): Testing the Scope

2021 ◽  
Author(s):  
Mullapudi Mohan Reddy ◽  
Pavankumar Janardhan Bhandari ◽  
Britto Sandanaraj
Keyword(s):  
Self Assembly ◽  
Serine Proteases ◽  
Rational Design ◽  
First Generation ◽  
Cysteine Residue ◽  
Protein Labeling ◽  
Site Specific ◽  
Artificial Proteins ◽  
Self Assembling ◽  
Biophysical Studies

Self-assembling artificial proteins (SAPs) have gained enormous interest in recent years due to their applications in different fields. Synthesis of well-defined monodisperse SAPs is accomplished predominantly through genetic methods. However, the last decade witnessed the use of few chemical technologies for that purpose. In particular, micelle-assisted protein labeling technology (MAPLabTech) has made huge progress in this area. The first generation MAPLabTech focused on site-specific labeling of the active-site residue of serine proteases to make SAPs. Further, this methodology was exploited for labeling of N-terminal residue of a globular protein to make functional SAPs. In this study, we describe the synthesis of novel SAPs by developing a chemical method for site-specific labeling of a surface-exposed cysteine residue of globular proteins. In addition, we disclose the synthesis of redox- and pH-sensitive SAPs and their systematic self-assembly and dis-assembly studies using complementary biophysical studies. Altogether these studies further expand the scope of MAPLabTech in different fields such as vaccine design, targeted drug delivery, diagnostic imaging, biomaterials, and tissue engineering.

scholarly journals Next Step in Gene Delivery: Modern Approaches and Further Perspectives of AAV Tropism Modification

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 750
Author(s):  
Maxim A. Korneyenkov ◽  
Andrey A. Zamyatnin
Keyword(s):  
Gene Therapy ◽  
Genome Organization ◽  
Rational Design ◽  
Structural Data ◽  
Rational Approach ◽  
Tissue Tropism ◽  
Adeno Associated Virus ◽  
Chemical Conjugation ◽  
The Usa ◽  
Therapy Delivery

Today, adeno-associated virus (AAV) is an extremely popular choice for gene therapy delivery. The safety profile and simplicity of the genome organization are the decisive advantages which allow us to claim that AAV is currently among the most promising vectors. Several drugs based on AAV have been approved in the USA and Europe, but AAV serotypes’ unspecific tissue tropism is still a serious limitation. In recent decades, several techniques have been developed to overcome this barrier, such as the rational design, directed evolution and chemical conjugation of targeting molecules with a capsid. Today, all of the abovementioned approaches confer the possibility to produce AAV capsids with tailored tropism, but recent data indicate that a better understanding of AAV biology and the growth of structural data may theoretically constitute a rational approach to most effectively produce highly selective and targeted AAV capsids. However, while we are still far from this goal, other approaches are still in play, despite their drawbacks and limitations.

scholarly journals Linking Ligand-Induced Alterations in Androgen Receptor Structure to Differential Gene Expression: A First Step in the Rational Design of Selective Androgen Receptor Modulators

2006 ◽  
Vol 20 (6) ◽  
pp. 1201-1217 ◽  
Author(s):  
Dmitri Kazmin ◽  
Tatiana Prytkova ◽  
C. Edgar Cook ◽  
Russell Wolfinger ◽  
Tzu-Ming Chu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Rational Design ◽  
Dynamic Structure ◽  
Rational Approach ◽  
Peptide Phage Display ◽  
Differential Gene ◽  
Receptor Modulators ◽  
Kinetics Of

Abstract We have previously identified a family of novel androgen receptor (AR) ligands that, upon binding, enable AR to adopt structures distinct from that observed in the presence of canonical agonists. In this report, we describe the use of these compounds to establish a relationship between AR structure and biological activity with a view to defining a rational approach with which to identify useful selective AR modulators. To this end, we used combinatorial peptide phage display coupled with molecular dynamic structure analysis to identify the surfaces on AR that are exposed specifically in the presence of selected AR ligands. Subsequently, we used a DNA microarray analysis to demonstrate that differently conformed receptors facilitate distinct patterns of gene expression in LNCaP cells. Interestingly, we observed a complete overlap in the identity of genes expressed after treatment with mechanistically distinct AR ligands. However, it was differences in the kinetics of gene regulation that distinguished these compounds. Follow-up studies, in cell-based assays of AR action, confirmed the importance of these alterations in gene expression. Together, these studies demonstrate an important link between AR structure, gene expression, and biological outcome. This relationship provides a firm underpinning for mechanism-based screens aimed at identifying SARMs with useful clinical profiles.

scholarly journals Labile disulfide bonds are common at the leucocyte cell surface

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110010 ◽  
Author(s):  
Clive Metcalfe ◽  
Peter Cresswell ◽  
Laura Ciaccia ◽  
Benjamin Thomas ◽  
A. Neil Barclay
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Disulfide Bonds ◽  
Reduction Process ◽  
Cysteine Residue ◽  
Surface Proteins ◽  
Protein Activity ◽  
Functional Changes ◽  
Reducing Conditions

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.

Designer drugs: pipe-dreams or realities?

Parasitology ◽  
1997 ◽  
Vol 114 (7) ◽  
pp. 145-151 ◽  
Author(s):  
W. E. GUTTERIDGE
Keyword(s):  
Chemical Synthesis ◽  
Rational Design ◽  
Molecular Design ◽  
Designer Drugs ◽  
Rational Approach ◽  
Parasitic Infections ◽  
Tropical Diseases ◽  
Track Record ◽  
Discovery Research ◽  
Long Run

New chemotherapies are urgently needed for the parasitic infections of animals and for the tropical diseases of man. Rational molecular design approaches to attempt to discover such products require a massive investment of resources up-front of actual chemical synthesis. However, such investment is justified, since chemical synthesis itself is highly resource-consuming. The fact that few targets have yet been validated to justify a rational approach is an argument only to get on and validate more. Not all the components of molecular design can yet be done totally rationally, but this is not an argument against applying this approach where it is possible. Absence of a successful track record is inevitable for any newly emerging technology. It is too early to draw conclusions about the relative costs of rational design versus empirical synthesis, since the former is only now beginning to become reality and the latter is in the middle of a (combinatorial) revolution. Similarly, it is too soon to predict with certainty which of these two approaches will prevail in the long run. However, they lend themselves to parallel tracks, so both may well continue for the foreseeable future. Current concerns about who would develop successful discoveries are not reasons for stopping discovery research. Indeed, a string of putative products held at the discovery/development interface would be useful ammunition to those trying to develop partnerships such as a Tropical Diseases R&D Alliance aimed at carrying out such work and sharing costs.

scholarly journals A Choline-Recognizing Monomeric Lysin, ClyJ-3m, Shows Elevated Activity against Streptococcus pneumoniae

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Dehua Luo ◽  
Li Huang ◽  
Vijay Singh Gondil ◽  
Wanli Zhou ◽  
Wan Yang ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bactericidal Activity ◽  
Rational Design ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Intraperitoneal Administration ◽  
Pharmacokinetic Analysis ◽  
Content Type ◽  
Equal Dose ◽  
Elevated Activity

ABSTRACT Streptococcus pneumoniae is a leading pathogen for bacterial pneumonia, which can be treated with bacteriophage lysins harboring a conserved choline binding module (CBM). Such lysins regularly function as choline-recognizing dimers. Previously, we reported a pneumococcus-specific lysin ClyJ comprising the binding domain from the putative endolysin gp20 from the Streptococcus phage SPSL1 and the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain from the PlyC lysin. A variant of ClyJ with a shortened linker, i.e., ClyJ-3, shows improved activity and reduced cytotoxicity. Resembling typical CBM-containing lysins, ClyJ-3 dimerized upon binding with choline. Herein, we further report a choline-recognizing variant of ClyJ-3, i.e., ClyJ-3m, constructed by deleting its C-terminal tail. Biochemical characterization showed that ClyJ-3m remains a monomer after it binds to choline yet exhibits improved bactericidal activity against multiple pneumococcal strains with different serotypes. In an S. pneumoniae-infected bacteremia model, a single intraperitoneal administration of 2.32 μg/mouse of ClyJ-3m showed 70% protection, while only 20% of mice survived in the group receiving an equal dose of ClyJ-3 (P < 0.05). A pharmacokinetic analysis following single intravenously doses of 0.29 and 1.16 mg/kg of ClyJ-3 or ClyJ-3m in BALB/c mice revealed that ClyJ-3m shows a similar half-life but less clearance and a greater area under curve than ClyJ-3. Taken together, the choline-recognizing monomer ClyJ-3m exhibited enhanced bactericidal activity and improved pharmacokinetic proprieties compared to those of its parental ClyJ-3 lysin. Our study also provides a new way for rational design and programmed engineering of lysins targeting S. pneumoniae.

scholarly journals A Dwarf Phenotype Identified in Breadfruit (Artocarpus altilis) Plants Growing on Marang (A. odoratissimus) Rootstocks

Horticulturae ◽  
2019 ◽  
Vol 5 (2) ◽  
pp. 40
Author(s):  
Yuchan Zhou ◽  
Steven J. R. Underhill
Keyword(s):  
Biochemical Characterization ◽  
Soluble Sugars ◽  
Primary Proton ◽  
Dwarf Phenotype ◽  
Standard Size ◽  
Tropical Fruit ◽  
Artocarpus Altilis ◽  
Two Parameters ◽  
Staple Crop ◽  
Reduced Activity

Breadfruit (Artocarpus altilis) is a tropical fruit tree primarily grown as a staple crop for food security in Oceania. Significant wind damage has driven an interest in developing its dwarf phenotype. The presence of any dwarf breadfruit variety remains unknown. Little is known regarding the growth of the species on rootstocks. Here, we examined the phenotype of breadfruit plants growing on marang (Artocarpus odoratissimus) rootstocks within 18 months after grafting; we identified a rootstock-induced dwarf trait in the species. This dwarf phenotype was characterized by shorter stems, reduced stem thickness and fewer branches, with 73% shorter internode length, 51% fewer and 40% smaller leaves compared to standard size breadfruit plants. The height of breadfruit plants on marang rootstocks was reduced by 49% in 9 months, and 59% in 18 months after grafting. The results suggest marang rootstocks can be applied to breadfruit breeding program for tree vigor control. Further biochemical characterization showed plants on marang rootstocks displayed leaves without change of total chlorophyll content, but with lower total soluble sugars, and stems with reduced activity of plasma membrane H+-ATPase, a well-known primary proton pump essential for nutrient transport. The significance of the two parameters in rootstock dwarfing is discussed.

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