Unveiling Protein Structure Dynamics Through Secondary Structure Rearrangement

Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Paper by Ramon K. Tabtiang, Brent O. Cezairliyan, Robert A. Grant, Jesse C. Cochrane, and Robert T. Sauer - Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Presented by Gayathri Priya Ravichandran

Structure Lot of basics Objective Introduction Methods Some discussions Reference Questions?

Basics Levels of protein structure Secondary Structure : The secondary structure of a protein describes certain repetitive, local conformations that are found in most peptide chains. The secondary structure does not describe the actual folding the protein in three dimensions, but instead illustrates the structure of small regions of the peptide. Primary Structure : The primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms

Levels of protein structure 1. 2. 3. 4. Primary amino acid sequence and disulfide bonds Secondary Local folding to form regular elements Tertiary 3 Dimensional folded structure Quaternary Tertiary structure of multimeric proteins

Alpha Helix It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding).

Beta Sheet The sheet (also -pleated sheet) is the second form of regular secondary structure in proteins. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A beta strand (also strand) is a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation.

Beta sheet

Operator In genetics, an operator is a segment of DNA to which a transcription factor binds to regulate gene expression. The transcription factor is typically a repressor, which can bind to the operator to prevent transcription. Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA (mRNA, tRNA or rRNA) by the enzyme RNA polymerase.

Repressor A repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers.

Transcription

Arc molecule Arc is a member of the immediate-early gene (IEG) family It is a rapidly activated class of genes functionally defined by their ability to be transcribed in the presence of protein synthesis inhibitors.

Homodimer A protein composed of two identical polypeptide chains. Polypeptides are chains of amino acids. Peptides are naturally occurring biological molecules. They are short chains of amino acid monomers linked by peptide (amide) bonds. A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules.

Objective Design a single-chain variant of the Arc repressor homodimer. Here? strands that contact operator DNA are connected by a hairpin turn. The? helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker, which is very stable

Arc repressor binding to DNA Tetrahelical bundle Beta sheets in contact with operator DNA

Objective The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature. A phenotype is the composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior (such as a bird's nest).

Introduction Consider a protein in which the ? helices and ? strands are disconnected but otherwise arranged properly in three dimensions, so that the hydrophobic core and most native interactions remain intact. Hydrophobic water fearing

Introduction There are probably many ways to connect the secondary structure elements that are compatible with stable folding. However, to our knowledge, no successful examples of noncyclic permutations of protein structural elements have been reported. ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)

Methods Molecular Biology. Constructed in a plasmid system by using a combination of PCR amplification of portions of the arc-st11 gene from pSA700. The structure of the final construct was verified by DNA sequencing. PCR Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies.

Methods Protein Purification. Uses E.coli for growing Isopropyl ? -D-thiogalactoside was added, and cells were harvested after an additional 2 h of growth. Processed using HCL, sodium phosphate and glycerol. The concentration of pArc was determined by absorbance using an extinction coefficient. Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.

Methods Crystallography. Crystals of pArc or selenomethionine- substituted pArc were grown at 20 C by hanging-drop vapor diffusion. Finally cycles of refinements are done to get isomorphous cell of the native crystal. Isomorphous having same crystalline form

Result - Structure

Results Design. As shown in Fig. 1C, the order of structural elements in single-chain Arc-L1-Arc is arm-? -? A-? B-linker-arm-? - ? A-? B (17). We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-? -linker-? -? A-? B-linker-? A- ? B (Fig. 1C). In pArc, unlike Arc-L1-Arc, the ? strands that contact operator DNA are now positioned together in the N- terminal part of the single-chain molecule, whereas the C- terminal portion comprises the tetra helical scaffold.

Results Expression and Structure. The pArc protein was expressed in E. coli It was soluble It was well behaved during purification.

Results Stability and Folding Kinetics. Rearranging the order of secondary structural elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation.

Results - Analytical Ultracentrifuge X-axis is called radial distance. It s a distance by which tubes are separated on centrifuge machines. Varying distance = varying force. Y axix is absorbance through spectrophotometer (measures density) -> corresponds to folding state of protein

Results - Denaturation Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar

Discussion Proteins are generally remarkably robust to amino acid substitutions, cyclic permutations, insertions, and even small deletions. The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate. However, not all cyclic permutations are tolerated, and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations.

Discussion The Arc fold was maintained after noncyclic rearrangement of its secondary structural elements, which required the addition of two non-natural linkers. It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc.

Discussion Previous studies did not find noncyclic permutants of barnase that folded like the wild-type protein, but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding. The protein design success mainly depends on the fold of the protein, the topology and the required linkers.

Reference Tabtiang, Ramon K., Brent O. Cezairliyan, Robert A. Grant, Jesse C. Cochrane, and Robert T. Sauer. "Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure." Proceedings of the National Academy of Sciences of the United States of America 102, no. 7 (2005): 2305-2309. Tsuji, Toru, Kenji Yoshida, Akira Satoh, Toshiyuki Kohno, Kensei Kobayashi, and Hiroshi Yanagawa. "Foldability of barnase mutants obtained by permutation of modules or secondary structure units." Journal of molecular biology 286, no. 5 (1999): 1581-1596. Lee, Cheolju, Michael P. Schwartz, Sumit Prakash, Masahiro Iwakura, and Andreas Matouschek. "ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal." Molecular cell 7, no. 3 (2001): 627-637. Wikipedia

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