amino acids and their properties - university...

Amino Acids and Their Properties

Recap: ss-rRNA and mutations

Ribosomal RNA (rRNA) evolves very slowly

Much slower than proteins

ss-rRNA is typically used

So by aligning ss-rRNA of one organism with that of another

We can estimate relatedness

Amino Acid Substitutions

Recall we can align DNA & RNA sequences

What does that mean?

We can also align two amino acid sequences

Can 2 nucleotides partially match?

Can 2 amino acids partially match?


Aligning sequences


Are some nucleotide mutations more significant than others?


Are some amino acid mismatches more significant than others?



Significance of a nucleobase mutation

Does name matter?

Does location matter?


Significance of an amino acid mutation

Name? Location?

Sequence matching and evolution rate

Proteins tend to evolve slower than DNA

Many DNA changes have no affect on a protein

A changed codon may map to the same amino acid

Non-coding DNA changes may have no effect

What does this mean for gauging the relatedness of

humans and chimpanzees?

humans and fish?




What might rRNA matching be good for measuring the relatedness of?

humans and chimpanzees?

humans and fish?

humans and what?





(what's that?)

However, different regions of ss-rRNA mutate at different rates

(Ribosome images next)

The Ribosome

Source: www.buzzle.com/articles/ribosomes-function.html

Ribosomes: diagrams and images

...check images.google.com for:

Ribosome diagram

Ribosome structure

Videos includehttp://www.youtube.com/watch?v=ID7tDAr39Ow

http://www.youtube.com/watch?v=ID7tDAr39Owhttp://www.youtube.com/watch?v=ID7tDAr39Ow

Recap: ss-rRNA and mutations




So by aligning ss-rRNA of one organism with that of another

We can estimate relatedness

Relatedness and Mutations

Much DNA mutates relatively quickly

Much ss-rRNA mutates relatively slowly

Much protein mutates at intermediate rates

Let's focus on protein mutation next

Amino acid subsitutions

Some amino acids substitutions are more likely than others

Why?

Amino acid substitutions

Some amino acids substitutions are more likely than others

Why?

Some are closer to others in terms of nucleobase codons

Some are closer in terms of resulting protein function

Amino acid substitutions II

Substituting similar ones is likely to Retain the protein structure and function

Substituting dissimilar ones is likely to Change the protein structure and function

Similarity of amino acids means what?

Amino acid substitutions III

Similarity of amino acids means similar physicochemical properties

Physicochemical: Concerning the physical and chemical Concerning physical chemistry

Physical chemistry: Connecting macroscopic properties of substances with their molecular

properties

Amino acid physicochemical properties

Nonpolar(Hydrophobic)

ACFGILMPVW

Polar (hydrophilic): NQSTY

Aromatic: FHWY (having to do with 6-carbon rings)

Basic: HKR

Acidic: DE (See http://www.bio.davidson.edu/courses/genomics/jmol/aatable.html By way of contrast, can anyone think of a non-

physicochemical property of some amino acids?

http://www.bio.davidson.edu/courses/genomics/jmol/aatable.htmlhttp://www.bio.davidson.edu/courses/genomics/jmol/aatable.html

Aromatic

Special type of ring-shaped molecule

Characterized by an unusual stabilizing property

Aliphatic

Non-aromatic

Amino acid abbrevs.

G=glycine, P=proline, T=threonine, A=alanine, , but why the following??

F=phenylalanine

Y=tyrosine

N=asparagine

Q=glutamine

W=tryptophan

Scoring protein sequence alignments

Simple way:

Two matching (identical) amino acids score 1

Two mismatching (non-identical) ones score 0

Goal: maximize % of matching amino acids

Works well for very similar sequences

Example:

CADQH

CADPM

Alignment score=___

Scoring protein sequence alignments II

Simple way ignores degree of similarity better to account for degree of similarity!

Solution: substitution matrices

PAM (Accepted Point Mutation, but PAM easier to say than APM) matrix Developed in 1970s by Margaret Dayhoff PAM1 matrix:

answers question, if 1% of the amino acids in a sequence change, at what rates would each amino acid be substituted for each other one?

Simple way ignores degree of similarity better to account for degree of similarity!

Solution: substitution matrices

PAM (Accepted Point Mutation, but PAM easier to say than APM) matrix Developed in 1970s by Margaret Dayhoff PAM1 matrix:



Substitution matrices PAM (Accepted Point Mutation, but PAM

easier to say than APM) matrix PAM1 matrix:


PAM2 matrix:

Not 2%! Rather, 1%, twice What is the difference?





PAM250 matrix:

Not 250%, obviously Why obviously? It is 1%, repeated 250 times!





PAM250 matrix:

It is 1%, repeated 250 times!

BLOSUM matrix is a popular type also

Scoring protein sequences: PAM250

Here is PAM250

source: http://bioinfo.cnio.es/docus/courses/SEK2003Filogenias/seq_analysis/PAM250matrix.gif

CADQH

CADPM

Alignment

score=?

http://bioinfo.cnio.es/docus/courses/SEK2003Filogenias/seq_analysis/PAM250matrix.gifhttp://bioinfo.cnio.es/docus/courses/SEK2003Filogenias/seq_analysis/PAM250matrix.gif

Scoring protein sequences: BLOSUM62 (default in Blast 2.0)

Source=http://bioinfo.cnio.es/docus/courses/SEK2003Filogenias/seq_analysis/pairwise.html.

Why do self substitutions have the highest numbers?

Why use PAM, BLOSUM, etc.?

Sequence similarity is related to evolutionary distance

Simple base matching (match/not) may work ok for closely related organisms humans and chimps, for example

Amino acid matching works better as evolutionary distance increases (why?)

Wed like to be able to assess relatedness of organisms that diverged long ago humans and worms, for example

Relatedness Long Ago

See images.google.com for

domains of life

We still are not sure, but the 3-domain system seems likely

But cladistics demands binary splits, so

3 domains requires 2 splits, and

2 domains are more related than the 3rd

Why use PAM, BLOSUM(II)

Organisms that diverged long ago have divergent analogous amino acid sequences

Since different amino acid substitutions occur at different frequencies we can measure relatedness back farther

e.g. when the fraction of identical amino acids is surprisingly low

and the fraction of identical base pairs is even lower

Comparing Sequences with PAMs (+ recap)

What does PAM mean?

PAM is considered an acronym for Point Accepted Mutation

Accepted Point Mutation (original)

Percent Accepted Mutations

A point mutation is a substitution of 1 amino acid for another

An accepted mutation is one that is passed down through the generations

Will a mutation be accepted if it is helpful? Harmful? Neutral? Helpful in some circumstances, harmful in others?

What Does PAM Mean, cont.

PAM has two meanings

PAM is a unit of evolutionary time

PAM is kind of substitution matrix

(The meanings are related)

PAM as a Unit of Time

A PAM is the amount of evolutionary change resulting in:

1 amino acid mutation per 100 amino acids

It is an average over >>100 amino acids

because mutations have randomness

After 1 PAM, will an organism have exactly 1% of its amino acids different from what they started out as?

PAM, Evolution, and Gaps

PAM ignores

Insertions

Deletions

Silent nucleotide substitutions (which are?)

PAM counts a change from A to B and back to A as 2 accepted point mutations

2 sequences 200 PAMs apart will have about 25% of amino acids the same!

PAM Matrices

They describe substitutability of amino acids, based on empirical evidence

Empirical = experiential

The matrices are derived from repositories of actual homologous sequences

A PAM 1 matrix is geared to best compare 2 sequences that are 1 PAM apart

A PAM 250 matrix is good for comparing quite diverged sequences

PAM 250 matrix is standard

Creating a PAM Matrix

Let fi be the frequency of amino acid i

We express fi as a fraction of the total

fi = instances of i __ .

instances of any amino acid

Frequencies range from

0.091 (L) down to 0.014 (W)

The most common amino acid occurs about ____ times more commonly than the least

Creating PAM matrix, cont.

Determine mutabilities of the amino acids

Some amino acids tend to change easily

Others not

If alanines mutability is set to 100

Serines mutability is 117 (highest, 1991 data)

Tryptophans mutability is 25 (lowest, 1991)

Lets look more closely at mi . . .


Mutability is a number

Given an evolutionary interval of 1 PAM

let mi = # mutations of amino acid i

# instances of amino acid i

Alternatively,

mi = p (an instance of i mutates)

Are the formulas on the previous slide identical?


Next, we break mi into constituent mi,js

That is, i mutates, but into j at what rate?

Use actual data from observed mutations

Populate a matrix of probabilities

The Diagonal

Values on the matrix diagonal do not really describe i mutating into itself!

(In reality, can that happen?)

They basically show

p (i does not mutate)

Thus, the columns add up to 1

Is the matrix on the last slide

Symmetric? Are there about 1% changed?

PAM0

What do you think a PAM 0 matrix might look like?

PAMn

Use matrix multiplication

PAM2 = PAM1 x PAM1

PAM3 = PAM2 x PAM1

PAM250? Do it 250 times!

PAM

What do you imagine a PAM matrix might look sort of like?

Logarithmicize

Actually, we take logarithms to get the usual matrix from the probability matrices

First, build another, reference matrix of expected probabilities

Assume all amino acids are equally mutable

Also assume they mutate into each other in proportion to their frequencies

(I.e., overall amino acid frequencies are maintained, but otherwise they dont care what they mutate into)

Logarithmicize

Now we have two matrices

Make a 3rd. Each entry is: Observed probability

Expected probability

were comparing reality to if mutations were truly random

Take the log of each entry to make a 4th An entry of 1 means 10x more mutations of that

type than expected

An entry of -1 means what?

Carrying On

We now use the matrix to measure relative evolutionary distance

amino acids and their properties - university...

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