Protein Design Research Papers - Academia.edu (original) (raw)

Results: No infusion was interrupted for symptoms, although 5 of 24 subjects had mild infusion-day adverse events thought to be possibly drug-related. No subject developed anti-FG-3019 antibodies. FG-3019 clearance was lower at 10 mg/kg... more

Results: No infusion was interrupted for symptoms, although 5 of 24 subjects had mild infusion-day adverse events thought to be possibly drug-related. No subject developed anti-FG-3019 antibodies. FG-3019 clearance was lower at 10 mg/kg than at 3 mg/kg, ...

Computational protein design (CPD) has established itself as a leading field in basic and applied science with a strong coupling between the two. Proteins are computationally designed from the level of amino acids to the level of a... more

Computational protein design (CPD) has established itself as a leading field in basic and applied science with a strong coupling between the two. Proteins are computationally designed from the level of amino acids to the level of a functional protein complex. Design targets range from increased thermo-(or other) stability to specific requested reactions such as protein–protein binding, enzymatic reactions, or nanotech-nology applications. The design scheme may encompass small regions of the proteins or the entire protein. In either case, the design may aim at the side-chains or at the full backbone conformation. Herein, the main framework for the process is outlined highlighting key elements in the CPD iterative cycle. These include the very definition of CPD, the diverse goals of CPD, components of the CPD protocol, methods for searching sequence and structure space, scoring functions, and augmenting the CPD with other optimization tools. Taken together, this chapter aims to introduce the framework of CPD.

Enzymes utilize substrate binding energy both to promote ground state association and to selectively lower the energy of the reaction transition state.i The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in... more

Enzymes utilize substrate binding energy both to promote ground state association and to selectively lower the energy of the reaction transition state.i The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the center of a 20 base-pair DNA target site, with the N-terminal domain of the enzyme making extensive binding interactions with the left (−) side of the target site and the similarly structured C-terminal domain interacting with the right (+) side.ii Despite the approximate two-fold symmetry of the enzyme-DNA complex, we find that there is almost complete segregation of interactions responsible for substrate binding to the (−) side of the interface and interactions responsible for transition state stabilization to the (+) side. While single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (−) DNA half-site almost exclusively increase KD and KM*, and those in the (+) half-site primarily decrease kcat*. The reduction of activity produced by mutations on the (−) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the utilization of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (−) side do so by modulating KM*, while redesigns with altered specificities on the (+) side modulate kcat*. Our results illustrate how classical enzymology and modern protein design can each inform the other.

Folding free energy of a protein is a delicate balance between stabi- lizing and destabilizing non-covalent itneractions. In this work, we decompose folding free energy into physically meaningful contributions, in which we aim to find... more

Folding free energy of a protein is a delicate balance between stabi- lizing and destabilizing non-covalent itneractions. In this work, we decompose folding free energy into physically meaningful contributions, in which we aim to find general trends. Empirical potential is used to calculate interaction ener- gy between all protein fragments, which are classified based on their dominant term in multipolar expansion. Calculations are done using 1200 non-redundant structures from PDB database. Based on general trends found in interactions between these fragments, we attempt to better understand relationships between interaction energies calculated using computational chemistry methods and their corresponding free energy contributions on stabilization.

Computational protein design (CPD), a yet evolving field, includes computer-aided engineering for partial or full de novo designs of proteins of interest. Designs are defined by a requested structure, function, or working environment.... more

Computational protein design (CPD), a yet evolving field, includes computer-aided engineering for partial or full de novo designs of proteins of interest. Designs are defined by a requested structure, function, or working environment. This chapter describes the birth and maturation of the field by presenting 101 CPD examples in a chronological order emphasizing achievements and pending challenges. Integrating these aspects presents the plethora of CPD approaches with the hope of providing a " CPD 101 ". These reflect on the broader structural bioinformatics and computational biophysics field and include: (1) integration of knowledge-based and energy-based methods, (2) hierarchical designated approach towards local, regional, and global motifs and the integration of high-and low-resolution design schemes that fit each such region, (3) systematic differential approaches towards different protein regions, (4) identification of key hot-spot residues and the relative effect of remote regions, (5) assessment of shape-complementarity, electrostatics and solvation effects, (6) integration of thermal plasticity and functional dynamics, (7) negative design, (8) systematic integration of experimental approaches, (9) objective cross-assessment of methods, and (10) successful ranking of potential designs. Future challenges also include dissemination of CPD software to the general use of life-sciences researchers and the emphasis of success within an in vivo milieu. CPD increases our understanding of protein structure and function and the relationships between the two along with the application of such know-how for the benefit of mankind. Applied aspects range from biological drugs, via healthier and tastier food products to nanotechnology and environmentally friendly enzymes replacing toxic chemicals utilized in the industry.

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights... more

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25°C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25°C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to ∼5%). It is demonstrated that a single universal mathematical function can be used to represent the partial molar heat capacity of the native and unfolded states of proteins in solution. This function can be experimentally written in terms of the molecular weight, the polar and apolar solvent accessible surface areas, and the total area buried from the solvent. This unique function accurately predicts the different magnitude and temperature dependences of the heat capacity of both the native and unfolded states, and therefore of the heat capacity changes associated with folding/unfolding transitions. © 1995 Wiley-Liss, Inc.

Ab initio in silico design of proteins and enzymes has emerged as a powerful tool to design application-tailored proteins and catalysts for a wide range of applications. Several enzymes exploit the unique features of metal cofactors to... more

Ab initio in silico design of proteins and enzymes has emerged as a powerful tool to design application-tailored proteins and catalysts for a wide range of applications. Several enzymes exploit the unique features of metal cofactors to achieve catalytic activity otherwise unattainable through the use of only natural amino acid residues. One of the major bottlenecks in ab initio design of novel proteins relies on long-range and epistatic effects that severely limit the possibility of a rational design. Within this framework there is an ongoing effort to reduce protein length and complexity to unlock the full potential of in silico protein design. In this work we specifically address this problem designing and investigating the dynamic features of 10 in silico designed minimal metallo-proteins. In particular, in this paper we investigate whether and to what extent it is possible to design a minimal metallo-enzyme made of only residues involved in metal binding. In this research we address these questions by investigating the ability of 10 different “mini-proteins” with a length shorter than 15 residues. Molecular dynamics studies clearly show that it is possible to design a minimal protein able to bind a metal atom with the correct geometry. It is noteworthy that designed mini-proteins cannot achieve the formation of a canonical hydrophobic core, rather the metal ion provides a “metal core” around which the entire protein is organized. This opens the possibility of designing synthetic enzymes composed of only functional residues organized around a “metal core” which acts as both structural and functional determinat.

Determination of the three dimensional (3D) structure of a protein can provide important details about its biological functions and mechanism of action. However, despite their significance, the precise three-dimensional structures of most... more

Determination of the three dimensional (3D) structure of a protein can provide important details about its biological functions and mechanism of action. However, despite their significance, the precise three-dimensional structures of most of the proteins are not fully determined till date. The main focus of the current review article is to gain a better understanding of the structural features of the proteins using computational techniques, and their relationship with function. Protein modeling and design is the method aimed to fold a primary amino acids sequence into protein structure with the ultimate goal of designing novel function and behavior. Moreover, proteins can also be designed from scratch or by similarity with the known protein structure. In the current article we have tried to cover various computer aided protein modeling and designing via homology and ab initio modeling, folding study using Molecular Dynamics (MD) methods and in silico mutation analysis.

Newborn Balb/c mice are highly susceptible to infection by the six coxsackievirus serotypes of group B (CVB) and it is known that receptor for these viruses are in highest concentration in the brain as compared to other tissues.... more

Newborn Balb/c mice are highly susceptible to infection by the six coxsackievirus serotypes of group B (CVB) and it is known that receptor for these viruses are in highest concentration in the brain as compared to other tissues. Therefore, proteins from the brain tissues of these animals were solubilized (Brain-Ext) and characterized for the identification of mouse brain receptor (MBR) proteins. Virus-blot analyses of Brain-Ext suggested that each of three virus variants of CVB3-(N, W and RD) recognized four receptor proteins designated p46, p44, p36 and p33 according to their molecular size. Similar analyses of cultured neurons from newborn Balb/c mice revealed the presence of the same four receptor proteins, while astrocytes appeared to possess only p46 and/or p44. Isoelectric focusing of Brain-Ext, focused MBR proteins in the pH range 4.0-8.5, with a peak around pH 5.7. P46 was found to be neuraminidase sensitive. A polyclonal rat antiserum (anti-MBR) protected cultured neurons and astrocytes against infection by CVB3, inhibited virus binding to these cells and recognized the same four receptor proteins on western-blots as detected on virus-blots by CVB3. However, a rabbit polyclonal anti-HeLa cell antiserum, which strongly binds to HeLa cells and protects them from CVB3 infection, neither recognized any of the receptor proteins in western-blot analyses of Brain-Ext nor inhibited CVB3 infection on cultured neurons and astrocytes. Conversely, anti-MBR did not recognize any of the receptor proteins by western-blot analysis of HeLa cell extracts nor did it inhibit CVB3 infection of HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Although the analysis and design of turns that connect the strands in antiparallel β-hairpins has reached an advanced state, much less is known concerning turns between antiparallel helices in helical hairpins. We have conducted an... more

Although the analysis and design of turns that connect the strands in antiparallel β-hairpins has reached an advanced state, much less is known concerning turns between antiparallel helices in helical hairpins. We have conducted an analysis of the structures and sequence preferences of two types of interhelical turns, each of which connects the two helices by a two-residue linker in an αL–β conformation. Based on this analysis, it became apparent that the turn introduced into a designed four-helix bundle protein, DF1, did not occur within an ...

An antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was... more

An antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5–6.2) and at low temperature (4–35°C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70°C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific deficiency of argininosuccinate synthetase (ASS) protein. We have recently identified the gene responsible for CTLN2, viz., SLC25A13, which encodes a... more

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific deficiency of argininosuccinate synthetase (ASS) protein. We have recently identified the gene responsible for CTLN2, viz., SLC25A13, which encodes a calcium-binding mitochondrial carrier protein, designated citrin, and found five mutations of the SLC25A13 gene in CTLN2 patients. In the present study, we have identified two novel mutations, 1800ins1 and R605X, in SLC25A13 mRNA and the SLC25A13 gene. Diagnostic analysis for the seven mutations in 103 CTLN2 patients diagnosed by biochemical and enzymatic studies has revealed that 102 patients had one or two of the seven mutations and 93 patients were homozygotes or compound heterozygotes. These results indicate that CTLN2 is caused by an abnormality in the SLC25A13 gene, and that our criteria for CTLN2 before DNA diagnosis are correct. Five of 22 patients from consanguineous unions have been shown to be compound heterozygotes, suggesting a high frequency of the mutated genes. The frequency of homozygotes is calculated to be more than 1 in 20,000 from carrier detection (6 in 400 individuals tested) in the Japanese population. We have detected no cross-reactive immune materials in the liver of CTLN2 patients with any of the seven mutations by Western blot analysis with anti-human citrin antibody. From these findings, we hypothesize that CTLN2 is caused by a complete deletion of citrin, although the mechanism of ASS deficiency is still unknown.