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Basic protein structure and stability II: Topics in side chain and backbone chemistry/ Basic anatomy of protein structure. Biochem 565, Fall 2008 08/27/08 Cordes. Covalent chemistry of the side chains and the backbone (main chain). reactions involved in enzymatic catalysis

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Basic protein structure and stability II:Topics in side chain and backbone chemistry/Basic anatomy of protein structure

Biochem 565, Fall 2008

08/27/08

Cordes

covalent chemistry of the side chains and the backbone main chain
Covalent chemistry of the side chains and the backbone (main chain)
  • reactions involved in enzymatic catalysis
  • covalent posttranslational modifications that regulate activity and processing of proteins, and also cause covalent damage
  • use of covalent side chain chemistry in analysis of proteins (e.g. crosslinking, attachment of labels, fluorophores etc)
  • Use of side chain and backbone chemistry in in vitro peptide chemistry

Both enzyme catalysis and posttranslational modifications will be covered later in the course. Some other important of the chemistry of side chains will also be covered in a class handout. In lecture today, we will cover just a few interesting, modern examples you may not hear about elsewhere.

chemistry of cys thiol group has been exploited in native chemical ligation ncl of peptides
Chemistry of Cys thiol group has been exploited in native chemical ligation (NCL) of peptides

The nucleophilic character of the thiolate anion of Cys can be used to effectively catalyze specific peptide bond formation between two peptides, if a Cys is present at the N-terminal end of one of the peptides, and if the other peptide is labelled with a thioester at its C-terminus.

The Cys thiol exchanges with the thioester, followed by S-N acyl transfer to the amine group of the Cys, forming a peptide bond.

The limitation of this reaction is that it requires Cys at the junction between the two peptides: you need an N-Cys peptide.

Tam et al Peptide Science60,194 (2001);

Muralidahran & Muir, Nature Reviews 3, 429 (2006)

protein splicing
translated sequence

of a protein

Protein splicing

N-extein

intein

C-extein

intein

N-extein

C-extein

splicing

intein

N-extein

C-extein

mature protein

excised intein

protein splicing mechanism
Protein splicing mechanism

N-X

acyl shift

transesterification

Asn cyclization

X-N acyl shift

and succinimide

hydrolysis

Perler & Adam Curr. Opin, Biotech. 11, 377 (2000)

protein splicing mechanism at a glance
Protein splicing mechanism at a glance

N-X

acyl shift

transesterification

Asn cyclization

X-N acyl shift

and succinimide hydrolysis

Perler & Adam Curr. Opin, Biotech. 11, 377 (2000)

asparagine deamidation
Asparagine deamidation

Asparagine deamidation is a major route of protein degradation and damage in vitro and in vivo. It is of concern with regard to the purity and proper function of peptide and protein pharmaceuticals.

adapted from

Xie L & Schowen, RL

J Pharm Sci 88, 8(1999)

See also Kossiakoff AA Science 240, 191 (1988)

oxidative damage modification of methionine

Oxidative damage/modification of methionine

Methionine oxidation is implicated in a variety of aging-related

disorders such as cataract formation in the lens and Alzheimer’s disease, though it may also serve nonpathological roles.

see for example Kantorow M et al. PNAS101, 9654 (2004;

Schoneich Arch Biochem Biophys 397, 302 (2002)

the basic anatomy of protein structure
The basic anatomy of protein structure

We will now discuss how we look at/describe/analyze protein

structures.

For Friday, I would like you to read the anatomy section of the web version of JS Richardson’s classic 1981 article the “Anatomy and Taxonomy of Protein Structure”, found at:

http://kinemage.biochem.duke.edu/~jsr/index.html

This version contains updated notes based on new structural insights since the original article.

the backbone conformation of proteins combination of regular and irregular structures
The backbone conformation of proteins:combination of regular and irregular structures

human

hexokinase

type I

(1QHA)

102 kDa

Rosano et al.

Structure 1999.

pig insulin

(1ZNI)

5.7 kDa

Bentley et al.

Nature 1976.

These “ribbon” diagrams show the “skeleton” of a protein. They are a smoothed representation of the “backbone” or “main chain” structure and do not show the side chains

slide14
Backbone or main-chain conformation

There are three bonds between “main chain” atoms (everything but the side chain) per residue, and torsional rotation can occur about any of these bonds, in principle. Hence, each residue has 3 angles that describe the main chain conformation for that residue.

backbone conformation resonance forms of the peptide group
Backbone conformation:resonance forms of the peptide group

The delocalization of the lone pair of the nitrogen onto the carbonyl oxygen shown in the resonance form on the right imparts significant double bond character (40%) to the peptide bond.

Breaking of this double bond character by rotation of the peptide bond requires on the order of 18-21 kcal/mol.

Consequently there is not free rotation around the peptide bond: rotation about the peptide bond happens on the time scale of seconds/minutes--very slow

consequences of double bond character in the peptide bond
Consequences of double bond character in the peptide bond

the peptide C-N bond is 0.12A shorter than the Calpha-N bond.

and the C=O is 0.02A longer than that of aldehydes and ketones.

All six of the atoms highlighted at left lie in the same plane, and as with carbon-carbon double bonds there are two configurations--cis and trans (trans shown at left)

slide17
Consequences of double bond character in the peptide bond

trans peptide bond

cis peptide bond

Still another consequence: in the cis form, the R groups in adjacent residues tend to clash. Hence almost all peptide bonds in proteins are in the trans configuration.

slide18
...and that means that the dihedral angle describing rotation around the

peptide bond, defined by the four atoms Ca(i)-C-N-Ca(i+1), will generally be close to 180°. This angle is known by the greek symbol w.

So the properties of the peptide bond place a strong restriction on the backbone conformation or main-chain conformation of proteins, that is to say, the spatial configuration of the non side-chain atoms.

slide19
The peptidyl-proline bond

cis peptidyl-proline

trans peptidyl-proline

• The peptidyl proline bond is an exception.

• It can be in either the trans or cis configuration, and the equilibrium constant favors the trans only very slightly.

• Roughly 20% of all peptidyl proline bonds in native proteins are in the cis configuration.

• This is in part because the amide hydrogen is replaced by a methylene group, which can clash with the R group of the preceding amino acid.

• Remember that there is still a kinetic effect on the rate of isomerization of the peptidyl proline bond that is similar to that for other peptide bonds--proline cis-trans isomerization can take seconds/minutes to occur, and this can actually limit the rate at which a protein adopts its “native” configuration beginning from a disordered structure (more on this later).

slide20
Backbone or main-chain conformation

So, one of the three degrees of freedom of the protein backbone is essentially eliminated by the properties of the peptide bond. What about the other two?

slide21
f and y angles

carboxy (C) terminal end

peptide planes

Torsional angles are defined by

four atoms:

y --> Ni-Ca-C’i -Ni+1

f --> C’i-1-Ni-Ca-C’i

Notice that these are defined

from N to C terminus, using

main chain atoms only.

A residue’s conformation is usually

listed as (f,y), since w is close

to 180 for almost all residues.

amino (N) terminal end

slide22
and between carbonyls

Unlike the peptide bond, free rotation occurs about the other two backbone bonds, but steric interactions within the polypeptide still severely limit plausible conformations, so that only certain combinations of phi and psi angles are “allowed”

or combinations of an R group, carbonyl or amide group.

between R groups

180

not allowed

0

allowed

-180

-180 0 180

steric clashes disallow some f and y combinations
Steric clashes disallow some f and y combinations

Theoretical calculations using hard sphere approximations suggest which phi and psi combinations cause clashes, and between which atoms.

Cross-hatched regions are “allowed”

for all residue types. The larger regions in the four corners are allowed for glycine because it lacks a side chain, so that no steric clashes involving the beta carbon are possible.

from web version of JS Richardson’s “Anatomy and

Taxonomy of Protein Structure”

http://kinemage.biochem.duke.edu/~jsr/index.html

observed f and y combinations in proteins
Observed f and y combinationsin proteins

Phi-psi combinations actually

observed in proteins with known

high-quality structures.

Gly residues are excluded from

this plot, as are Pro residues and

residues which precede Pro

(more on this later).

Contours enclose 98% and 99.95%

of the data respectively.

Notice that the observed conformations

do not exactly coincide with the

theoretically allowed/disallowed confs

based on steric clashes.

from Lovell SC et al.

Proteins 50, 437 (2003)

sample ramachandran plot for a protein
red= allowed

yellow= additionally allowed

pale yellow= generously allowed

white= disallowed

Sample “Ramachandran plot” for a protein

The squares denote non-glycine

residues, while the triangles are

glycines. Glycines have no side

chain and are not as restricted

because of the lack of side chain

steric clashes.

This protein has 219 residues,

90% of which are in the “allowed”

region and 10% of which are

in “additionally allowed” regions.

None are in the other regions

(except glycines,

which don’t count)

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