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115 Lecture 2
the EF hand and Calmodulin
20
Biochemistry
Undergraduate 1
04/29/2013

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Term
a m...... is a conserved amino acid sequence alignment: it is a local alignment corresponding to a region whose f............ or s........... is known, or its significance may be unknown
Definition
motif, function, structure
Term
the EF hand is a protein motif that is associated with the binding of which divalent cation?
Definition

ca2+

 

it was named from the ca2+ binding site that occurs between the E and the F alpha helices in parvalbumin

Term
what configuration is the EF hand characyerised by?
Definition

HELIX-LOOP-HELIX configuration.

 

the motif is thought to resemble a clenched right hand. there are >150 EF hand containing proteins. Members of this superfamily are found soley in the cytosol

Term

EF hands bind calcium with an affinity of 

KD ~ 10-6 M

in the presence of 3mM mg2+

 

what is Kd?

Definition
it is the disassociation constant where 50% of molecules are actively bound.
Term
what is the extracellular, intracellular (unstimulated) and intracellular (stimulated) levels of calcium?
Definition

extracellular 10-3

intracellular (unstimulated) 10-7

intracellular (stimulated) 10-5/-6

Term
what does the presence of a glycine at position 15 and isoleucine at position 17 of the EF loop permit?
Definition

glycine at position 15 permits a sharp bend in the loop

isoleucine at position 17 attaches the lop to the hydrophobic core of the molecule.

Term

how many residues does the EF hand contain?

 

which amino acid is found in position 1 of the first alpha helix?

Definition

29 residues.

 

the first alpha helix has a Glu at position 1

 

it also has hydrophobic residues facing the core of the molecule at postions 2,5,6, and 9 thus, the alpha helices are amphipathic

Term
similarly the s....... alpha helix has a Glu at postion 21. Residues 22, 25, 26 and 29 are h............
Definition
second, hydrophobic
Term
although 6 amino acids are involved in calcium binding, there is an octahedral environment of their arrangement. How many oxygen atoms are ligands?
Definition

seven. Six oxygens atoms from Calmodulin and one of water

 

in the octahedral environment the glutamic acid at -Z acts as a bidentate ligand. The calcium is contained in a tight loop and interacts with the acidic amino acids. acidic amino acids have carboxylate oxygen ligands

Term
camcium is coordinated by the side chains of f.... amino acids that can be assigned to the vertices of an octhedron.
Definition

five.

 

the sixth amino acid has calcium coordinated by a peptide carbonyl

Term
to summarise the EF hand is a protein motif of 29 amino acids.
Definition
its function is to bind calcium ions tightly once calcium ion levels reach around 10-6 M
Term
name the calcium modulating protein that binds calcium and is almost made up entirely of EF hands?
Definition

Calmodulin

 

 

changes in the intracellular calcium ions are important signals in all eukaryotic cells

Term
calcium2+ is an important s............. m............ involved in muscle contration. it controls the release of h........... and n............. It is involved in binding of carbohydrates by l........... Many ca2+ binding proteins have a common structural motif known as the EF hand. This motif is formed from t.... helices linked by a turn
Definition
second messenger, hormones, nuerotransmitters, lectin, two
Term

how many EF hands does calmodulin contain?

what is the Mr of calmodulin?

Definition

it contains 4 EF hands

it is a 17kDa protein with a highly conseved amino acid sequence.

 

the KD of  calmodulin is about ~10-6 M

Term
why does calmodulin need calcium to be bound to it?
Definition

so it can bind to and activate various kinases. these are molecules that add phosphate groups to other molecules.

 

the resting intracellular calcium level is 10-7 M but when Calcium is acting as a second messenger, the conc rises briefly to between 10-6 M and 10-5 M

Term

what kind of shape does a calmodulin protein have?

 

what structure seperates the N-terminal from the C-terminal?

Definition

a dumbell shape.

 

the N-terminal domain has 2 EF hands seperated from a similar C-terminal domain by a unique 6-turn single alpha helix.

Term

what does calcium binding trigger in calmodulin?

 

what sort of patch is exposed as a result of this trigger?

Definition

a conformational change.

 

when 3-4 calciums ions bind to calmodulin, a conformational change is triggered that exposes a hydrophobic patch in each globular domain.

Term
the hydrophobic patch is exposed by the molecule folding at which region, amino acid and postion?
Definition

in the central region

amino acid: Serine

Position: 81

 

these hydrophobic patches can interact with other proteins

Term
when binding to a target peptide what does the long helix of the dumbell do?
Definition

it unwinds and the two lobes of calmodulin swing round to enfold the alpha helical target.

 

a hydrophobic patch on each lobe of the calmodulin contacts the hydrophobic side of the helical target peptide. Thus, the target peptide sits in a hydrophobic channel.

it can only do this with the shape that happens as a result of calcium binding.

Term
target enzymes for calmodulin include Phosphorylase kinase, myosin light chain kinase, adenylate cyclases and the Ca2+ ATP-ase
Definition

Target enzymes have a mixture of basic and hydrophobic amino acids which are capable of adopting an alpha helix.

 

Calmodulin is a sensor of rises in intracellular calcium and calcium enhances its affinity for a number of important regulatory proteins. the calcium/calmodulin system is part of a signal transduction pathway.

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