Protein splicing

a molecular process for the removal of undesired regions of protein from pre-pro form to final translational protein

1. Protein splicing
analogous to mRNA splicing
k. k. gupta

2. INTRODUCTION
•Protein is an array of amino acids .
•Once synthesized , it may have some intervening
sequences which are not needed in the final protein
product .
•So it has to be removed from the pre pro protein form.
•Protein splicing is a process of removal of these intervening
/ interrupted sequences .
•These intervening sequence is called as Intein.
•It is just similar to post transcription process so it is better
referred as post translational process .
•After translation, a protein is heterogenous/splitted
consisting alternate two segments like HnRNA .These are :-

3. •Aninteinis a segment of aproteinthat is able to excise itself and
join the remaining portions (theexteins) with apeptide bond
•.Inteinshave also been called "proteinintrons". The concerned
protein splicing process is thus called as Intein-mediatedProtein
splicing occurs after the intein-containingmRNA has
beentranslatedinto a protein.
•This precursor protein contains three segments—anN-
exteinfollowed by the inteinfollowed by aC-extein.
•After splicing has taken place, the resulting protein contains the N-
exteinlinked to the C-extein; this splicing product is also termed an
exteinwhich determine the structure and function as per folding
state..
Synthesized protein
EXTEIN
INTEIN/Intervening sequence of protein

4. Splitted Protein
DNA

5. Potential inteinreactions
. Protein splicing results in ligation of the N-extein (E N ) and C-extein (E C
When inteins are mutated or inserted in heterologous contexts, are unable to splice.
off-pathway reactions can occur resulting in N-terminal, C-terminal, or double
cleavage products that are unable to splice. Off-pathway N-terminal cleavage can
occur in both the linear and the branched (thio)ester intermediates. Off-pathway C-
terminal cleavage occurs when cyclization of the intein C-terminal residue precedes
branch intermediate formation.
Splicing requires sequential nucleophilic displacement reactions
catalyzed by strategies similar to proteases and asparagine lyases.

6. Types of intein
•Based on their domain structure, inteinsare categorized in four
classes:
•full-length inteins,
•mini-inteins,
•split-inteinsand
•alanine-inteins.
•Most inteinsare full-length inteinsexpressed within a single
polypeptide chain.
•They are bifunctionalproteins that include two structural domains -
the inteindomain responsible for protein splicing out of the
precursor polypeptide chain, and a homing endonuclease(HE)
domain with a role in DNA-cutting and insertion of the associated
mobile genetic element into the precursor protein-coding gene
The HE domain splits the
splicing domain into N and C-
terminal splicing domains.

7. Splicing domain
•The splicing domain consists of conserved blocks A, B, F
and G, while blocks C, D, and E are present in the HE
domains. Blocks C and E contain conserved endonuclease
active sites with catalytic residues Asp, Gluor Lys
•Blocks A, and B, localize near the N-terminus of the intein, while blocks F and G are
located near the C-terminus Conserved amino acids important for the splicing
process (Cys, Ser or Thr) are present both at the inteinN-terminus as well as on the
C-exteinfragment near the intein-exteinjunction

8. Types of intein

9. •For example when first inteinwas discovered in
1988 through sequence comparison of ATPase
gene of Neurosporacrassa(with Intein) and
carrot vacuolar (without intein) it was found that
final product is similar i.eInteinhas been
removed from the neurospora.
•A similar homologous gene in yeast (with intein)
was also described called putativecalcium ion
transporter protein.

10. Mechanism
•autocatalytic and naturally process i.einteinitself
mediate splicing. involved bond transfer and no input
of energy .\
•chemically generate medium-sized proteins called
native chemical ligation from the long sized pro protein
.This reaction requires specific amino acids sequence
which is located at the junction of extein-inteinand
involve three nucleophilesas splicing domain which
are conserved in such pro-protein
•Ser, Thror Cysat the inteinN-terminus
•Asnor Glnat the inteinC-terminus; and
•Ser, Thror Cysat the beginning of the C-extein

11. Steps I: N-O shift
• N-O shift –A typical inteinsbegins with ser or cysand ends in Asn.
•The first residue in the C exteinis ser, thr/cys.
•These residues often function as nucleophiles.
•A nucleophilesare those which is electro-negative (Nucleophiles) and can attract
electropositive nucleus atom.
•For example in this case ser of C exteinhas R group represented as CH
2OH i.eOH
in which O is electro negative which attack on N of As. i.eN-O shift.
•It is the transition of peptide bond between the aaend of the inteinand n-extein
in to an ester or thioesterbond, these transtitiondepends on a nucleophilesattack
of the bond by the side chain of ser or cysresidues at the amino terminal end of
intein(-OH or SH respectively .
•This reaction is called N-O when the attacking atom is O and N/S &N and when it
is sulphercalled N-S shift

12. Class 1 inteins with a C-terminal Asn and a Cys,
Ser, or Thr at the first position in both the intein
and the C-extein splice using the standard four-
step protein splicing mechanism .
X represents an oxygen or a sulfur atom. For clarity, tetrahedral
intermediates and residues facilitating each step are omitted.
Although the definition of an intein is the excised sequence

13. Trans esterification
•In this step the side chain of the first residue
of the C extein attacks the ester /thioester
bond at the amino end of the intein .
•Here too, the attack is by a polar side chain of
a ser/ thr or cys.
•This leads to a trans esterification and
formation of thioester bond between N
extein and C extein

14. 3
rd
steps -Asn cyclisation
•cyclisationof the Asnside chain leads to cleavage
of the peptide bond between the inteinand the c
extein(c terminal splice junction).
•This reaction removes inteinfrom the ligated
exteinwhich are linked together at the ester
bond .
4
th
steps O-N shift–This step of protein
splicing is spontaneous and is the reverse N-O/N-S
shift taking place and peptide bond formation occurs
between N-C exteins

15. Mechanism
anucleophileis a
chemical species that
forms bonds
withelectrophilesby
donating an elctronpair
. Allmolecules and ions
with a free pair of
electrons or at least one
pi bond can act as
nucleophiles. Because
nucleophilesdonate
electrons, they
areLewis bases.