Cell and Molecular Biology Aug 22, 2009 Protein Structure and Function 06/02/myosin2.jpg Assignment reading:

Cell and Molecular Biology Aug 22, 2009

Protein Structure and Function

http://biosingularity.files.wordpress.com/2006/02/myosin2.jpg

Assignment reading: chapter 3 and journal paper

'Glowing' jellyfish grabs Nobel

Jellyfish will glow under blue and ultraviolet light because of a protein in their tissues. Scientists refer to it as green fluorescent protein, or GFP.

Fluorescence protein is part of the gene---cells constantly emits green fluorescence

Fluorescence protein has been bounded to protein inside the cells---fade within weeks in the absence of antifading mounting media

Brainbow

http://news.bbc.co.uk/2/hi/science/nature/7658945.stm

Glowing mouse

The monomer unit of protein: amino acid

Panel 3-1 Cell and Molecular Biology, 4th edition

Formation of peptide bond

Figure 3-1. Cell and Molecular Biology, 4th edition

Proteins form by condensation of amino acids in a reaction that releases water, is coupled to ATP hydrolysis and is catalyzed by enzymes within the ribosome.

Amino acids are commonly joined together by an amide linkage (peptide bond)

Figure 3-4 Cell and Molecular Biology, 4th edition


A protein consists of a polypeptide backbone with attached side chains. Protein differs in its sequence and number of amino acids. The two ends of a polypeptide are the amino terminus (N-terminus) and the carboxyl terminus (C-terminus). The amino acid sequence of a protein is always presented in the N-to-C direction, reading from left to right.

There are 20 amino acids

It is useful to remember their most important structural features.At a minimum, memorize their names and one letter codes.

Know which one is acidic, basic, hydrophobic, polar, big, small, reactive, inert

There are a few modifications done to amino acids as or immediately after the protein is made:e.g. phosphorylation, acetylation, acylation, glycosylation


Essential amino acids

The side chains



Peptide sequence


Three types of noncovalent bonds that help proteins fold. Although a single one of these bonds is quite weak, many of them often form together to create a strong bonding arrangement. The final folded structure, or conformation, adopted by any polypeptide chains is the one with the lowest free energy.

Non-covalent bonds in protein folding


Formation of hydrogen bonds


Large numbers of hydrogen bonds form between adjacent regions of the folded polypeptide chain and help stabilize its three-dimensional shape. The protein depicted is a portion of the enzyme lysozyme, and the hydrogen bonds between the three possible pairs of partners have been differently colored, as indicated.

The polar amino acid side chains tend to gather on the outside of the protein, where they can interact with water; the nonpolar amino acid side chains are buried on the inside to form a tightly packed hydrophobic core of atoms that are hidden from water. In this schematic drawing, the protein contains only about 30 amino acids.


Proteins have hydrophobic cores

Changes in protein conformation


Some proteins, often small ones, reach their proper folded state spontaneously. Once unfolded, kT allows them to find their equilibrium structure when returned to physiological conditions. Other proteins are metastable: they are helped to fold to structures they would practically never find at random. Protein folding in a living cell is often assisted by special proteins call molecular chaperones.

A protein can be unfolded, or denatured, by treatment with certain solvents to disrupt the non-covalent bonds or heat (heat denaturation) and cold (< 20C for certain antibodies)

Only a very small fraction of random sequences of amino acids make polymers with a unique or stable structure. Nature has selected those sequences with specific folded shapes. The shapes and therefore functions can be very fragile to even tiny changes in atomic structure (mutation). A single protein can have separate sections each with its own folded domain, and linked by spacers.


http://www.ks.uiuc.edu/Research/fibronectin/

Disulfide bond covalently link polypeptide chains together, providing a major stabilizing effect on a protein.

Disulfide bonds stabilize protein structure



Different levels of protein structure

Non-covalent bonding stabilizes protein folding, which can be categorized by 4 different levels of structure.

Primary structure: linear amino acid sequenceSecondary structure: regular orientation due to H-bond -helix, -pleated sheetTertiary structure: full 3-D organization of a polypeptide chainQuaternary structure: multi-subunit complex consisting of multiple polypeptide chains

Large proteins (~ 50-2000 amino acid long) come in a wide variety of shapes, generally consisting of several distinct protein domains—structural units that fold more or less independently of each other.

The regular conformation of the polypeptide backbone observed in the α helix and the β sheet. (A, B, and C) The α helix. The N–H of every peptide bond is hydrogen-bonded to the C=O of a neighboring peptide bond located four peptide bonds away in the same chain.

Secondary structure: alpha helix


Two (or three ) α-helices can wrap around each other to form a stable coiled-coil structure. By doing so, both helices have most of their nonpolar (hydrophobic) side chains lined up on one side so that they can twist around each other with these side chains facing inward. This long coiled-coil structural framework for many elongated proteins such as -keratin, myosin, and collagen.

http://www.bio.miami.edu/~cmallery/255/255prot/gk2x37.coil.gif

Alpha helices can form a stable coiled-coil structure


(D, E, and F) The β sheet. In this example, adjacent peptide chains run in opposite (antiparallel) directions. The individual polypeptide chains (strands) in a β sheet are held together by hydrogen-bonding between peptide bonds in different strands, and the amino acid side chains in each strand alternately project above and below the plane of the sheet


Secondary structure: beta sheet


Beta sheets can have parallel or antiparallel strands

Protein domains, e.g. Src protein


A single polypeptide can have more than one independently folded domain. This feature is exploited in nature and biotechnology to make chimeric proteins.

It’s not yet possible to predict the folded shape from amino acid sequences. Good predictive rule for 2nd structure but not for the higher level structures.

The two conformations are strikingly similar as well as their amino acid sequences (green) and the location of active sites (red). However, these active sites have different enzymatic activities to cleave different peptide bonds. The difference is due to the genetic modification (copies of genes) during evolution.

Proteins can be classified into many families

Family of serine proteases (digestive enzymes)


The present-day proteins can be grouped into protein families. Each family member has amino acid sequences and 3-D conformation resembles those of the other family members while performing different functions.

Function within a structure: protein modules

Protein module (~ 40-200 amino acids) is a subset of protein domain that has a versatile structure as found in a variety of different contexts in different molecules.

Protein modules provide a convenient framework for the generation of extended structure. The in-line arrangement with N- and C- terminal in opposite ends can readily link in series to form extended structure either with themselves or with other molecules.



Any region of a protein’s surface that can interact with another molecule through sets of non-covalent bonds is called a binding site.

The tight binding of two folded polypeptide chains at this site creates a larger protein molecule which a precisely defined geometry. Each polypeptide chain in such a protein is called a protein subunit.


Two identical subunits bind head-to-head, held together by a combination of hydrophobic forces (blue) and a set of hydrogen bonds (yellow region).

Large protein molecules contain more than one polypeptide chain


A long chain of identical protein molecules can be constructed if each molecule has a binding site complementary to another region of the surface of the same molecule. Therefore, protein molecules can assemble to form filaments that may span the entire length of a cell.

www.bio.miami.edu/~cmallery/255/255prot/255proteins.htm

Hierarchy in protein assembly

GlobularFigure 3-24

HelixFigure 3-26

FiberFigure 3-28

Final shape (3 most common conformations)

Protein: classified by functions

Enzymes catalytic activity and function (-ase)

Structural collagen of tendons and cartilage, keratin of hair and nails

Transport proteins bind and carry ligand

Motor proteins can contract and change the shape of cytoskeleton

Defensive antibodies, thrombin

Regulatory growth factors, hormones, transcription factors

Receptor cell surface receptors

Protein Function: How shape determins function?

The specific binding of protein molecules determines their activity and function--- 3-D shape/conformation matters. Binding always shows great specificity.

Many weak bonds are needed to enable a protein to bind tightly to a second molecule, which is called a ligand for the protein. A ligand must therefore fit precisely into a protein's binding site, like a hand into a glove, so that a large number of noncovalent bonds can be formed between the protein and the ligand.




Protein conformation determines its chemistry. • arrangement of neighboring parts of the polypeptide chain to exclude water molecule• the clustering of neighboring polar amino acid side chains can alter their reactivity

Enzymes

• Enzymes are very important class of proteins that determine all the chemical transformations that make and break covalent bonds in cells• Enzymes bind to one or more ligands, called substrates, and convert substrates to products.• Enzymes may speed up reaction without themselves being changed---catalysts.


Enzymes accelerate chemical reactions by decreasing the activation energy.

Allosteric enzymes: feedback mechanism

Many enzyme has at least two different binding sites on their surfaces:

active site--- recognizes the substrate regulatory site--- recognizes regulartory molecule

Interaction depends on a conformational change in the protein: binding at one of the sites causes a shift from one folded shape to a slightly different folded shape.

Figure 3-57. Cell and Molecular Biology, 4th edition Figure 3-58. Cell and Molecular Biology, 4th edition

positive regulation negative regulation

Many protein functions are driven by phosphorylation

Phosphorylation regulates thousands of protein functions in a typical eukaryotic cells. Phosphorylation occus by the addition of a phosphate group to amino acid side chains, usually the OH- terminal of serine, threonine and tyrosine. This cause a major conformational change in protein due to charge interaction or the attached phosphate group becomes part of the structure.


Protein kinases: catalyze phosphorylation (addition of phosphate)

Proten phosphatases: catalyze dephosphorylation (removal of phosphate)


Individual protein kinases serve as microchips.

Cyclin-dependent protein kinase (Cdk) regulates the cell cycle.

Cdk becomes active when:1.Cyclin is present2.Pi added to specific threonine side chain3.Pi removed from tyrosine side chainWhen all 3 requirements are met, Cdk is turned on.

GTP binding proteins as molecular switches


The activity of a GTP-binding protein (also called a GTPase) generally requires the presence of a tightly bound GTP molecule (switch “on”). Hydrolysis of this GTP molecule produces GDP and inorganic phosphate (Pi), and it causes the protein to convert to a different, usually inactive, conformation (switch “off”). As shown here, resetting the switch requires the tightly bound GDP to dissociate, a slow step that is greatly accelerated by specific signals; once the GDP has dissociated, a molecule of GTP is quickly rebound.

Phosphorylation in cell signaling

Many signaling pathways important for the cell survival involve GTP-binding proteins (GTPases). The phosphate group is part of GTP that binds very tightly to the protein it regulates. When the tightly bound GTP is hydrolyzed to GDP, this domain undergoes a conformational change that inactivate it.


GTP = molecular switch

Ras protein important role in cell signaling


The large conformational change in GTP-bound domain


EF-Tu Ras protein

The addition and dissociation of phosphate group causes a shift of a few tenths of a nanometer at the GTP binding site. This tiny movement causes a large conformational change to propagate along the switch helix. The helix serves as a latch that adheres to a specific site in another domain of the molecule. After GTP hydrolysis, the switch helix detaches, allowing the two domains to swing apart over a distance of ~ 4 nm.

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Cell and Molecular Biology Aug 22, 2009 Protein Structure and Function 06/02/myosin2.jpg Assignment reading: