Protein Folding Protocols (Methods in Molecular Biology Vol 350)

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Amylases are employed in various industries to multiply functions for example it is used in food industry for softening bread, adjusting flour for liquefaction and scarification of starch as well as juice treatment. In detergent and paper industry, these enzymes are frequently used to eliminate starch stains and de-inking For the production of certain food and industrial products starch is converted into bioethanol or into food ingredients like fructose, glucose and organic acid s in microbial fermenters, requiring biocatalysts such amylase for the liquefaction and scarification.

Thus to improve the activity and stability of amylases at harsh conditions both protein engineering and DNA recombinant technology are being frequently used. Rice has been well reported as an instance for the production of industrial useful biocatalysts from raw material of agriculture Lipases are also used intensively by food and detergent industries such as for lipid stain removal, chees flavor, dough stability and as contaminants controller in paper and pulp industry.

For food processes toxicologically safe lipases are required which are obtained from Candid arugose. Different commercial isoforms of lipases are produced by DNA shuffling, computer modeling and protein engineering Later on a comprehensive study was accomplished on mutagenesis and protein engineering to enhance the catalysis of microbial lipases Applications referring to remediation of polluted environments oxygenases, laccases and peroxidases are three major classes of enzymes, which have significant role in environmental applications for biodegradation of organic and toxic pollutants.

But mostly, these enzymes face problems like enzyme denaturation by toxic compounds, inhibition of ES enzyme-substrate complex and low catalytic activity. Scientists have done intensive work to overcome these problems by developing engineered enzymes by recombinant technology and rational enzyme design Medical and clinical applications: Protein engineering has vast number of applications in the area of therapeutics.

Formerly protein engineering is accomplished to achieve second generation recombinant protein having considerable properties in medical and clinical applications Mutation, DNA shuffling and recombinant DNA approach were used in protein engineering to get superior results of therapeutic protein Afterward up-gradation in protein engineering led to fabrication of secreted therapeutic proteins, namely, interferon, insulin, etc.

Up-gradation of therapeutics for combating against cancer is the major area of interest in protein engineering. One of latent treatment recommended for cancer is pre-targeted immunotherapy in which radiation toxicity is noticed to be minimized. By using protein engineering, the application of this pre-targeted immunotherapy was anticipated to be a competent treatment for cancer Up-gradation in recombinant DNA technology and protein engineering facilitates the synthesis of novel antibodies that can be successfully applied as anti-cancer drugs. These distinctive antibodies are engineered such a manner that they specifically recognize and strongly associated with their cancerous antigenic markers and assist in eliminating the cancerous cell with greater precision.

Development in protein engineering leads to some of its other noteworthy medical applications. One of them is protein cationization technique, which assists in development of future therapeutics Tissue regeneration and polymer based drug delivery system was another major target of protein engineering Targeted drug delivery remains the important feature of a novel biopharmaceutical to attain successful therapies. Functional proteins and peptides are engineered with an efficient carrier for sufficient and targeted delivery of drug in this apprehension.

Certainly, health care can be more operational if the diagnosis is speedy, accurate and perceptive. Nearly genetic disorders have been reported so far. The majority of human contain a few genes without any sign of disease and many of them are accountable for susceptibility, however molecular basis of majority of these diseases is still unclear. Successful efforts have been made in last more than three decades in sighting into diagnosing genetic disorders prior to embryonic implantation in humans and credited a lot of merit.

Beadle and Tatum anticipated one gene-one hypothesis, was condemned later has shown that certain genes consequence in dozens of proteins 64 , probably get produced either in traces with a very short half-life, splitted, chemically changed or the fragments of different genes may be reorganized. For that specific reason, under such physiological conditions gene analysis is not suitable in clinical diagnosis of the proteins and eventually proteomics desires the characterization of certain proteins that are key agents of a cell and gene products. These agents straightforward contribute to the drug development as all drugs are directed against proteins, except a few, get in the way in DNA replication in cancer cells and RNA in AIDS virus multiplicity.

Thus, advancements in protein detection and characterization protocols would assist in diagnosing diseases with accuracy and sensitivity. Hereafter, up-gradation in protein nano-technologies having been carried out in recent years, is comprehensively updated here. It is fairly noteworthy to monitor the protein concentration in a biological sample prior to investing for its practical biological activity. The accurate estimation of less abundant protein is the prime challenge, having been overcome by evolution of nano-technology Fluorometric assay 68 , ELISA 69 , radioimmunoassay 69 and immunofluorosence 69 measurement tools are evolved to quantify the proteins in nano quantity and even less, although, except spectrofluorometric 68 technique, those are multi-step, difficult and rather time-consuming techniques.

Protein engineering in nano-biotechnology: The applications of protein engineering in nano-biotechnology are moving ahead with the time. Nanotechnology was not receiving substantial credit for their difficult synthesis and assembly in functional systems. Then after, a phase came with the studies on biomolecular structural organizations revealing their hierarchical arrangements from nano to macro levels. Proteins, lipids and carbohydrates are the biological macromolecules, being used for biosynthesis of tissues under synchronized gene expression s. Proteins are the most noteworthy amongst them as they are the structural constituents during tissue formation and aid to the transport and arrangement of building blocks and accessories.

Therefore proteins are the major focus for nano-technological systems in their synchronized synthesis and assemblage. The combinatorial tools of biology used in protein engineering such as the technologies of bacterial cell surface display and phage display also get their applications in nanobiotechnology to monitor selectively binding polypeptide sequences to inorganic surfaces. Individual clones, likely to be specific in their binding to an inorganic material surface are principally revealed through stepwise washings of phages or cells in the biological method named as bio-panning.

Sequencing of these clones is performed in view of obtaining the amino acid sequences of these polypeptides, purposely bind to semi-metal oxides and other nano-technology surfaces. Nano-biotechnology did extremely well further through another technique employing Genetically Engineered Proteins for inorganics i. Subsequently, a number of specific peptides, being bound to certain surfaces like quartz and gold, have been selected and characterized 70 , Besides, computational methods were combined with experimental approaches in view of better engineering the binding of peptides followed by accurate assembly of nano-technology systems revealing superior function specific peptides that can be used in therapeutics, tissue engineering and nano-technologies employing biological, organic and inorganic materials Protein engineered peptides are employed in biosensors, used as molecular motors and transducers, in the generation of biocompatible nano-materials.

Bioinformatics analyses have also great impact in this emerging field of protein engineering Amyloid fibrils are also attractive application of protein engineering in the construction of nano wires as they provide as the templates. In fact, this is a characteristic of many of the proteins that they figure an organized aggregate of fibrils, namely, amyloid fibrils. This salient feature of well-organized non-covalent aggregate formation ability of amyloid fibrils directs their use in nano-technology with self-assembly and organization of small molecules being quite specific and vital Pioneering proteins recognized as affibody binding proteins, being of non-immunoglobulin Ig origin have been developed employing protein engineering techniques.

They have high affinity and thus are potentially considered in diagnostics, viral targeting, bio-separation and tumor imaging as well 73 , For development of novel biosensors for analytical diagnosis, insertional protein engineering has been noticed to immerge during a decade 1 , The amino acid succession and organization in a protein affects its conformation as well as function.

Consequently, the capability to transform the sequence and thus the structure and activity, of entity proteins in a methodical fashion, explore many opportunities, both scientifically and for exploitation in bio-catalysis. Modern techniques of synthetic biology, whereby increasingly large sequences of DNA can be synthesized de novo , allow an incomparable ability to engineer proteins possessing novel functions.

Enzymologists differentiate binding K d and catalytic k cat stages. In a similar manner, judicious approaches have blended design for binding, specificity and active site modeling with more empirical methods of classical directed evolution DE for improving k cat where natural evolution rarely pursues the highest values , principally with respect to residues distant from the active site and where the functional linkages supporting enzyme dynamics are both unknown and hard to predict.

The aim of this overview is to bring to light some of the approaches, being developed to allow using directed evolution for improving enzyme characteristics, often noticeably. It has been registered that directed evolution varies in a various ways from natural evolution, including in picky the accessible mechanisms and the potential selection pressures. Therefore, it is hereby firmly focused on opportunities afforded by techniques, which enable protein engineer or enzymologist to map sequence to structure and activity in silico , as an effective ways of modeling and thus exploring protein landscapes.

As identified landscapes may be assessed and rational about as a whole, concurrently, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape triangulation, experienced by sequence-activity interactions and joined to the promising techniques of synthetic biology, offers scope for the development of novel biocatalysts that are both extremely dynamic and strong.

Further, for gene expression analysis, zinc finger protein engineering is becoming fascinating for molecular biologists. Afterward a three-finger protein was effectively engineered to study the expression of an oncogene in mouse cell line 75 , The understanding of gene regulation and structure and function of the human genome improved dramatically at the end of the 20th century.

Conversely, the technologies for manipulating the genome have been slower to develop. For example, the arena of gene therapy has been focused on correcting genetic diseases and increasing tissue repair for more than four decades. Though, with the exception of a few very low efficiency techniques, conformist genetic engineering approaches have only been competent to supplement auxiliary genes to cells. This has been a substantial complication to the clinical success of gene therapies and has also intended for severing inadvertent concerns in several cases.

Consequently, technologies that make possible the defined modification of cellular genomes have diverse and notable implications in many facets of research and are noteworthy for translating the products of the Genomic Revolution into perceptible benefits for medicine and biotechnology. To address this requirement, in s, a task was embarked to expand technologies for engineering protein-DNA interactions with the rationale of generating custom tools competent of targeting any DNA sequence.

The objective has been to let researchers to reach into genomes to specifically control, knock out, or replace any gene. To realize these aims, it has principally been focused on understanding and manipulating zinc finger proteins. Specifically, it is required to create a simple and straight forward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins employing only defined modular components, a web-based usefulness and standard recombinant DNA technology. Two substantial challenges faced so far were i The development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and ii Determining how individual zinc finger domains could be chained together as polydactyl proteins to identify exclusive locations within complex genomes.

Various researchers have since employed this modular assembly technique to engineer artificial proteins and enzymes, which activate, repress or make definite changes to user-specified genes in human cells, plants and other organisms. Besides, they engineered certain novel techniques for externally regulating protein activity and delivery have been successfully developed 76 , as well as developed certain new approaches for the directed evolution of protein and enzyme function.

However, in biofuel industry, to obtain biofuels from lignocellulosic materials, such cellulose enzymes are produced by protein engineering, which have improved catalytic activity and reduced the production costs of biofuels Protein cysteine modification, an approach of protein engineering, produces proteins with diverse functions 78 , The usage of proteins as therapeutics has a long history and is becoming ever more common in modern medicine.

Despite the fact that number of protein-based drugs is growing every year, major problems still remain with their application. Among these complications are quick degradation and excretion from patients, consequently requiring recurrent dosing that in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to improve these problems is to link a polyethylene glycol PEG group to the protein of interest.

This procedure called PEGylation has grown strongly in recent years occasioning in several approved drugs. Installing a single PEG chain at a definite site in a protein is quite challenging. There has been substantial research into several approaches for the site-specific PEGylation of proteins.

After introducing the site-specific PEGylation, recent developments using chemical methods have been comprehended. That is followed by a more extensive discussion of bio-orthogonal reactions and enzymatic labeling. More specifically, such novel proteins are frequently used to develop new therapeutic proteins, which show improved half-life and reduced toxicity 78 , Protein engineering is one of the applications of recombinant DNA technology.

Rational design requiring the prior knowledge, has gained significance because of computational algorithms and techniques generating useful output from protein sequence. Directed evolution on the other hand is a extensive process concerning screening and selection but provides a fair possibility to have protein that might not be present in nature.

Though conventional techniques have always been confirmed valuable, protein engineering has contributed to study functional properties in more varied way. Classes of engineered enzymes such as proteases and amylases have substantial applications in food, detergent, paper and several other industries. Other classes such as peroxidases and oxygenases are being applicable in environmental studies. Pharmaceutical products such as engineered antibodies have also been in market. Novel engineered proteins are being used in diagnostics and biosensors.

Bibliographic Information

Besides, nano-biotechnology is also receiving benefit through this field. Protein Engineering will keep on as a source for creating diversity in proteins to be used as experimental tools in metabolic engineering and protein studies. Author is grateful to Prof. Subscribe Today. Science Alert. Principal events in the evolution of amino acid synthesis: The way amino acid s are synthesized has altered during the history of Earth. The hadean eon represents the time from which Earth first formed. The subsequent Archean eon about 3, million years ago is known as age of bacteria and archaea.

All Rights Reserved. Review Article. Singh and Abbas Ali Mahdi. Similar Articles in this Journal. Search in Google Scholar. Singh and Abbas Ali Mahdi, American Journal of Biochemistry and Molecular Biology, 9: DOI: Adrio, J. Demain, Microbial enzymes: Tools for biotechnological processes.

Biomolecules, 4: Rehman, M. Tariq and S. Chen, Development of therapeutic proteins: Advances and challenges. Chang, G. Lee and J. Shaw, Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce. Food Chem. Muzaffar, M. Awan, S. Din, I.

Protein Folding Protocols (Methods in Molecular Biology) - AbeBooks:

Nasir and T. Husnain, Genetically modified foods: Engineered tomato with extra advantages. Life Sci. Abbas, Rehman, A. Iqbal, S. Din and A. Rao et al. Nanotechnology, a new frontier in agriculture. Golotin, A. Podvolotskaya and V. Rasskazov, Genetically modified proteins: Functional improvement and chimeragenesis. Bioengineered, 6: Megeed, M.

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Casali, K. Rege and M. Yarmush, Engineering protein and peptide building blocks for nanotechnology. Rice and P. Daugherty, Rapid isolation of high-affinity protein binding peptides using bacterial display. Protein Eng. Boche, H. Gille and U. Brinkmann, Development of secreted proteins as biotherapeutic agents. Expert Opin.

Protein engineering of subtilisin. Yuan and H. Zhao, Recent advances in DNA assembly technologies. Fessner, G. Sprenger and A. Samland, Recent progress in stereoselective synthesis with aldolases. Swainston, P. Day and D. Kell, Synthetic biology for the directed evolution of protein biocatalysts: Navigating sequence space intelligently. Beadle's progeny: Innocence rewarded, innocence lost. MacCallum, However, it has yet to make a significant progress in protein folding. In a recently published work, a specially designed super computer succeeded in the folding of two small proteins Shaw, Maragakis et al.

Although computing power does not seem to be the greatest hurdle from now on, this success is unlikely to extend broadly in the near future. The greater challenge lies in the accuracy of simulation force fields which will be discussed later. In this chapter, we focus on theoretical studies of protein folding by molecular dynamics simulations. The kinetics of protein folding can be studies by conventional molecular dynamics CMD.

But the insufficient sampling in current CMD simulations prevents the extraction of thermodynamic information. This has prompted the development of enhanced sampling techniques, among which the most widely adopted technique is replica exchange molecular dynamics REMD , otherwise called parallel tempering. In the past few years, we have applied both CMD and REMD to the ab initio folding — meaning folding from extended polypeptide chain without any biased force towards the native contacts — of several model proteins, including villin headpiece subdomain HP35 , B domain of protein A BdpA , albumin binding domain ABD , and a full sequence design protein FSD.

The accuracy of protein folding reached sub-angstrom in most of these simulations, a significant improvement over previous simulations. Based on these high accuracy simulations, we were able to investigate the kinetics and thermodynamics of protein folding. The summary of our findings will be presented here in details. Finally, we will stress the critical role of force filed development in studying folding mechanism by simulation.

chapter and author info

Villin headpiece subdomain HP35 is a small helical protein 35 residues with a unique three helix architecture Fig 1. The three-dimensional structure was solved earlier by an NMR experiment and more recently by a high resolution X-ray experiment Chiu, Kubelka et al. Due to the small size and rich structural information, HP35 has attracted a lot of attention from both experimentalists and theoreticians. The productive folding always went through the major intermediate state while no productive folding was observed through the minor intermediate state.

On the other hand, Gly11 was likely most accountable for the flexibility of helix I. In addition, the high occupancy of short-distance native contacts and low occupancy of long-distance native contacts pointed to the importance of local native contacts to the fast folding kinetics of HP The folding landscape of HP35 was partitioned into four thermodynamic states, namely the denatured state, native state, and the two aforementioned intermediate states. A major free energy barrier 2. In addition, a melting temperature of K was predicted from the heat capacity profile, very close to the experimentally determined melting temperature of K.

Because of the small size, HP35 has been considered as a classical two-state folder. This notion is supported by some earlier folding experiments. However, our simulation clearly pointed to the existence of folding intermediates. Our two-stage folding model is supported by some more recent folding experiments. In a solid-state NMR study, three residues Val9, Ala16, and Leu28 from the three helices exhibited distinct behavior during the denaturation process, and a two-step folding mechanism was proposed.

In an unfolding study using fluorescence resonance energy transfer, Glasscock and co-workers demonstrated that the turn linking helices II and III remains compact under the denaturation condition Glasscock, Zhu et al. In a mutagenesis experiment, Bunagan et al. A recent freeze-quenching experiment by Hu and co-workers revealed an intermediate state with native secondary structures and nonnative tertiary contacts Hu, Havlin et al.

These experiments are highly consistent with our observations in terms of both the stepwise folding and the rate-limiting step. Kubelka et al. Therefore, the intermediate state lies on the folded side of the major free energy barrier, which is consistent with the separation of the unfolded state from the other states in our folding simulation. The estimation of 1. Nevertheless, controversy still exists regarding the folding mechanism of this small protein.

In a recent work by Reiner et al. It should be noted that different perturbations to the system, including high concentrations of denaturant, high temperatures, and site mutagenesis, have been utilized in different folding experiments. Because of the small size of HP35, the folding process may be sensitive to some of these perturbations. With the continuous development of experimental techniques that allow minimal perturbation and monitoring of the folding process at higher spatial and temporal resolution, the protein-folding mechanism will become more and more clear.

REMD is one of the most efficient sampling techniques for protein folding. However, due to the non-physical transitions from the exchange of conformations at different temperatures, its usage is mostly restricted to thermodynamics study. Consistent with REMD, the folding free energy landscape displayed four folding states Fig 2 , the denatured state on the upper right region, the native state on the lower left region, the major intermediate state on the lower right region, and the minor intermediate state on the upper left region.

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  • All five trajectories were combined together, and the population of each conformation in a small zone was converted to free energy by log transformation. From the folding landscape, we can see focused sampling in the native state, sparse sampling in the minor intermediate states, and heterogeneous sampling in the denatured state and the major intermediates state.

    The heavy sampling in the denatured state was likely due to the limited simulation trajectories. Ideally, thousands of trajectories are needed to reach good sampling. However, long simulations like this one are computer intensive beyond the capacity of a typical institution. The above-described 2D landscape is only an overall display of the conformational sampling.

    To get more details, we performed conformational clustering based on the combined five trajectories.

    A Review on Conventional and Modern Techniques of Protein Engineering and their Applications

    We here use the top ten most populated conformational clusters to describe the conformational sampling Fig 3. The center of each conformational cluster was used to represent the cluster.


    Among the top ten clusters, we can see three conformations in the native state clusters 2, 3 and 10, colored in purple , three conformations in the major intermediate state clusters 5, 6 and 9, colored in green , and four conformations in the denatured state clusters 1, 4, 7 and 8, colored in blue , while the minor intermediate state did not show up due to small overall population. The overall energy enthalpy was not a good indication of the folding.

    In fact, the energy of the native state conformations was the highest and that of the denatured state conformations was the lowest. Entropy evaluation has long been a difficult subject in the field of computational biochemistry. A breakthrough will extend the application of force fields to protein structure prediction.

    Kinetics and Thermodynamics of Protein Folding

    Based on conformational clustering, we can study the kinetics and thermodynamics of protein folding using a new technique called network analysis. Traditionally, protein folding is illustrated by 1D profiles such as RMSD global or partial , energy, solvent accessible surface area, radius of gyration and selected distances. The hyper-dimensional nature of protein folding makes none of these 1D profiles adequate to reflect the folding process.

    Arthur Horwich (Yale/HHMI) Part 1A: Chaperone-assisted protein folding

    The emergence of 2D maps such as the one in Fig 2 greatly alleviate the problem by combining two independent profiles in one map. However, 2D maps are still insufficient to represent the hyper-dimensional process. Under this circumstance, several novel approaches have been applied to protein folding in recent years, including the disconnectivity graph by Karplus and network analysis pioneered by Caflisch Krivov and Karplus ; Caflisch Network analysis has gained popularity in protein folding recently Bowman, Huang et al.

    In network analysis, protein conformations are represented as nodes and the transitions among different conformations are represented as edges. Both nodes and edges can be colored based on a specified property, and analysis can be done based on the topological distribution of conformations with a specified property. In the folding network of the combined five trajectories Fig 4 , we painted the nodes according to the state identity of the conformation and displayed the structure of the top ten populated conformations.

    From this network, we can see the clear separation of the denatured state from the native state and major intermediate state. The minor intermediate state was also connected to the denatured state. These findings were consistent with the observation from the 2D maps. A new finding is the mixing of the native state and major intermediate state which were clearly separated in the 2D map. The implication of this new finding is that the barrier between these two states is so small that they can easily convert to each other, which is supported by experimental evidence.

    This study demonstrated the power of network analysis and suggested more caution on interpreting 2D maps of protein folding. The global folding network better reflect the thermodynamics of protein folding. To understand the kinetics of protein folding better, a simplified network with shortest path can be constructed Fig 5. In this network, the shortest path connecting the denatured state, the major intermediate state and the native state was extracted from the global network.

    A clear flow of conformational transition from the denatured state to the major intermediate state and then to the native state was demonstrated in this network. Even the number of transitions between any neighboring conformations can be labeled on the network. In the denatured state, there were three short paths from the four top conformations to the major intermediate state, suggesting multiple folding pathways.

    Two conformations in the minor intermediate state were embedded in the denatured state, suggesting them as off-pathway intermediate. In the major intermediate state, the two top conformations close to the denatured state clusters 6 and 9 in Fig 3 had wrongly folded segment A, while the top conformation close to the native state cluster 5 in Fig 3 had a near native structure. This information on the intra-state conformational transition is also helpful to reveal the details in the protein folding process.

    Buy eBook. Buy Hardcover. Buy Softcover. FAQ Policy. About this book In this updated and expanded second edition of an established classic, the editors have added critical reviews to a fresh collection of cutting-edge protocols for gene expression in bacteria, fungi, plants, plant cells, animals, and animal cells. Show all.