Judie Berlier is a Staff Scientist at Thermo Fisher Scientific Inc., USA. Since joining Molecular Probes and continuing through acquisitions and mergers to become a Member of Thermo Fisher Scientific, one constant has been finding and using the technology needed to make novel and useful detection tools for cell biology.

Researchers want labeled antibodies that work in their specific applications. Traditional labeling methods include amine-reactive chemistry, thiol-reactive chemistry; periodate oxidation and carbodiimide reactions. Antibodies labeled with these approaches give stable conjugates but can also disrupt antigen binding and are difficult to characterize. In the worst of cases, antibody specificity is lost upon conjugation. A modular copper-less click method for antibody labeling has been shown to be both reproducible and robust and is currently the best option for antibodies where standard chemistries have failed. Antibodies from different sub-classes and species have performed remarkably well in a variety of applications when using this gentle approach. Additionally, this approach allows for the labeling of any existing antibody to be carried out in the presence of BSA, a common stabilizing protein included in the formulation buffer of many commercially available primary antibodies. The method developed uses UDP-GalNAz and the permissive enzyme beta-galactosyltransferase (GalT (Y289L) to produce a site-selective azide label on the N-linked glycans of the heavy chain Fc region far from the antigen binding domain. The azide tagged antibodies are reacted without metal catalysts with any dibenzocyclooctyne (DIBO)-functionalized probe of choice. These biologically unique moieties are inert and stable until the eightmember strained ring reacts with the azide modified antibody forming a stable triazole linkage. This site-specific universal labeling of IgG’s without genetic engineering can be applied to essentially any existing antibody and is currently the preferred method for reproducibly making well-defined antibody conjugates.

Ruud Van Deursen has completed his PhD from University of Berne. After Postdoctoral studies at Ecole Polytechnique Federale de Lausanne in Switzerland on highthroughput screening, he has joined the Baker Laboratory at the University of Washington in Seattle to work on de novo design of nucelophilic catalysts and expression of disulfide-rich proteins. He has published 16 publications as an author and co-author in the fields of CADD and biocatalysis

VX and analogous organophosphorous (OP) compounds display their toxicity by rapidly inhibiting acetylcholinesterases (AChE) in the central nervous system. Upon binding of the OP compound to the catalytic serine, AChE and similar naturally occurring enzymes are frequently reported to undergo ‘aging’. In this process the enzymatic activity is inhibited by irreversible binding of OP compounds to the histidine in the catalytic site. The membrane protein phospholipase A2 (Group VIII) is an exception and is widely resistant against aging, allowing the use of oximes as strong nucleophile in a competitive substitution reaction to restore the catalytic activity. Rosetta has been previously used to create new enzymes based on phosphotriesterases (PTE) obtained from soil bacteria. Further optimization of the catalytic efficiency is however needed. The well conserved α, β-hydrolase-type fold of the phospholipase A2 (group VIII) has been chosen as starting point of this alternate de novo design approach for the reason of the slow aging rates. Key part of this 60 residue minimal core is a flat beta sheet that is composed of 3 parallel strands with a length of 5 residues each. This sheet is surrounded by at least three alpha helices. The small size of this design asks for the introduction of disulfide bonds for stabilization. Herein we present our experimental setup to secrete these de novo designed disulfide-rich proteins using fusion constructs with different leader sequences of the Sec-, SRP- and TAT-secretion pathways.

Marcin Lukasz Ura has completed his PhD from Braunschweig University of Technology and he is currently a Postdoctoral Scholar in Tony Kossiakoff Laboratory at the Department of Biochemistry and Molecular Biology of the University of Chicago.

Lack of adequate structural information on Chromosome Passenger Complex (CPC) proteins is mainly attributed to their inherent disordered structures. Thus, there is a significant drive to dissect the structural information that will provide valuable insights into the biological function of these proteins. To cater to the growing demands of tools as probes to delineate the structure and function of the CPC proteins, we generated highly specific, synthetic affinity binders for the different domains of these proteins by phage display using a synthetic Fab library. The selected sABs were very specific for Survivin’s N-terminal Baculovirus Inhibitor of Apoptosis-repeat (BIR) domain for the N, C-terminal domain of Borealin as well as for the N-terminal domain of INCENP. High specificity of sABs was evident from ELISA-assays, Dot-blots and Western-blots. Immunofluorescence studies show typical intracellular localization of the sABs bound to the mitotic spindle, the nucleus and the midbody in dividing cells. Interestingly, sABs against the C- and N-terminal domains of Borealin reveal unique cellular patterns not reported to date. These sABs are also validated as customized primary reagents for Western-Blot. The generated high quality antibodies will be used as crystallization chaperones for structure determination of these candidates that are recalcitrant to crystallization by conventional approaches.

We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their terminal. The designed peptide displayed salt resistance and small toxicity to hRBCs and human fibroblast. Fluorescence microscopic studies indicated that the FITC-labeled peptide preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with designed peptide. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics.

Sriram Jakkaraju is currently pursuing PhD in Hornlab at Northern Illinois Univeristy. His PhD dissertation focuses on understanding thermodynamic orgins of enhanced affinity and its implementations on library based protien engineering.

A frequent goal of library-based antibody engineering is affinity maturation. Due to the complex nature of protein-protein interactions, little is known regarding the thermodynamic origins of enhanced affinity for such engineered proteins. Consequently, there is interest to understand the common energetic trends that drive affinity maturation. The resulting knowledge can be used to more efficiently direct library design and in vitro selection strategies. Here we explore the thermodynamic origins of an in vitro affinity matured anti-RNase A VHH Antibody (VHH-mat) that possesses an approximate 100-fold increase in affinity when compared to the parent antibody, VHH-wt. ΔΔΔG values of nine relevant interface residues were calculated from alanine scanning mutagenesis, which revealed four of nine residues were critical for VHH-mat’s increased in affinity. Thermodynamic binding and stability studies were conducted on individual, double and triple mutants as well as isolated CDR1 or CDR3 loop variants, where wild-type residues were replaced with the VHH-mat residues. These studies revealed the enhanced binding energy (~3 kcal/mol) of VHH-mat was primarily through non-additive contributions (~70% non-additive vs. ~30% additive). Interestingly, non-additive contributions correlated with the addition (positive) or removal (negative) of likely salt-bridge interactions between CDR3 residues. When comparing inter-CDR loop interactions (CDR1 and CDR3), only additive contributions were observed. The observed thermodynamic signature of VHHmat’s enhanced binding appears to directly follow the nature in which CDR1 and CDR3 were initially randomized individually during affinity maturation before combining together, thus greatly diminishing chances for inter-CDR loop non-additive contributions. These findings reveal the important role that localized, intramolecular interactions may have on enhanced affinity. Furthermore, the detailed dissection of the binding thermodynamics may help shape library design with the goal of developing affinity matured antibodies.

Gujjarlapudi Deepika has completed her MD Biochemistry from Osmania Medical College and presently pursuing her PhD in Asian Institute of Gastroenterology, Hyderabad India.

Mucopolysaccharidoses are a group of inherited lysosomal storage disorders (LSDs) consisting of seven distinct clinical types and numerous subtypes. These are the result of deficiency of certain lysosomal degradative enzymes which are required to breakdown glycosaminoglycans (GAGs). The clinical features observed among MPS subtypes show overlapping signs and symptoms with other LSDs and rheumatologic disorders. This makes clinical diagnosis a challenge. India has witnessed an increase in the burden of lysosomal storage disorders in the recent years. Aim of the study was the temporal pattern of the disease from onset of symptoms to the final diagnosis. Retrospective analysis of the case record forms of the patients attending the hospital over a period of 5 years was under taken only the data of the patients who had confirmatory enzyme analysis or mutation study for LSD was further analyzed. The age at onset, suspicion of the illness, first clinical presentation to a tertiary hospital with genetics and the age at the final diagnosis of these confirmed cases were considered. A total of 480 patients were referred to the genetic clinic in this period. The diagnosis of LSD was suspected in 232 patients (9.08% of all referrals) and it could be confirmed in 119 cases (22.37% of the suspected cases). Maximum patients were diagnosed with Gaucher disease (31.93% of the diagnosed cases) followed by Mucopolysaccharidoses (20.16% of the diagnosed cases. Mutational analysis was available in 22 patients (18.48% of the diagnosed cases. With the advent of new therapies, appropriate medical management is possible and hence establishing timely diagnosis has become crucial. In the retrospective data analyses done in our institute two different diagnostic approaches were discussed. The diagnostic approach includes GAG quantification followed by Two Dimensional cellulose Acetate Electrophoresis and enzyme analysis which seems to be appropriate for diagnosis MPS I, II, IV, VI and VII. The second diagnostic approach involves direct enzyme analysis which is pertinent for MPSII as these patients have a distinct clinical phenotype which enables clinician to establish the diagnosis with ease. The relative frequency of MPS I, II, IIIA, IIIB, IIIC, IVA, IVB and VI observed in our experience is 14%, 8%, 19%, 16%, 4%, 27%, 3% and 9% respectively. If family history of LSD is known initially, the pregnant women with gestation period between 16-22 weeks can be tested for GAG quantification in Amniotic fluid. With these two method combined it will be a useful, fast and cost effective diagnostic tool.

Myeongsang Lee has completed his Bachelor’s degree from Department of Mechanical Engineering, Dongguk University and completed his Doctoral studies in Korea, Department of Mechanical Engineering; Korea University, South Korea. He is majoring in protein engineering, especially in computational protein engineering using molecular dynamics. He has published 5 papers in reputed SCI (E) journals.

Amyloid protofibrils are mainly implicated to degenerative and neurodegenerative diseases such as type-2 diabetes, dialysis related diseases, Alzheimer’s disease and Parkinson’s diseases and so forth. Pathological characteristic of amyloid protein are not easily degraded under physiological condition which amyloid fibrils have high material properties. Based on these material characteristic, several research groups utilized amyloid fibrils as basic functional material template, which can be used in conductive nanowires, durg delivery carrier and nano structured biofilms etc. Recently, in vitro studies have been conducted to investigate the structural reaction and characteristic of amyloid proteins via force spectroscopy methods such as atomic force microscopy (AFM) and optical tweezers. However, computational methods are effectively revealing the mechanical properties of the amyloid protofibrils and provide structural information about the amyloids. In the present study, we reported the diverse material properties and behaviors based on different stacking and facing of polymorphic hIAPP segments using MD simulations and steered molecular dynamics (SMD) methods. From our results, we examined how these mechanical properties may differ with respect to the structural composition of beta strand segments.

Sneha Chandrakant Sawant is currently pursuing PhD Biotechnology from DBT-ICT Centre for Energy Biosciences; Institute of Chemical Technology, India. She has completed her Master of Science in Biotechnology from Mumbai University, India. Her area of research revolves around studying mechanistic behavior, structure- function relationships and protein engineering of enzymes involved in lignocellulose biomass depolymerization.

Product inhibition of β-glucosidase is a rate limiting step in saccharification of cellulosic biomass. Till now, the exact mechanism of either inhibition or in some cases activation of β-glucosidase by glucose is very elusive. Herein we investigated the structural basis for glucose tolerance by comparing glucose tolerant β-glucosidases with a β-glucosidase from Paenibacillus polymyxa, Glu1C that has lower glucose tolerance. Comparative sequence alignment based on 3-D structure was carried out between glucose tolerant β-glucosidase (PDB: 3AHZ) from Neotermes koshunensis and β-glucosidase from Paenibacillus polymyxa (PDB: 2O9T). In silico studies suggested the importance of active site residues: L174, G373 and W412. Among these residues mutability for W412 was the highest as based on evolutionary relationships. In order to understand the role of W412, two charged mutations of opposite charges were created in previously cloned and characterized Glu1C gene of β-glucosidase and expressed in E. coli DH5α. Biochemical characterization of these two mutants revealed that the first mutant W412E was only 33% active in comparison of Glu1C and other mutant W412R was marginally active. A two fold increase in thermal stability was found in case of W412E (t1/2=30 mins) at 50° C. However, the mutation resulted in marginal decrease in glucose tolerance as oppose to wild type. Hence, W412 residue, despite not being a part of catalytic residues, it was found that it was crucial to maintain optimal activity, thermal stability and glucose tolerance. Overall, presumbaly, β-glucosidase has a very delicate arrangements of structural elements that govern catalysis, stability and glucose tolerance.