Proteins
chapters 3~6
Virtually Everything for executing
biological phenomena!!!!
Chemical structure [Chapt 3]
Three dimensional structure [Chapt 4] Conformation
Function [Chapt 5]
Storage/Transport, Hemoglobin--oxygen-binding proteins
Defense, Immune system; immunoglobulins
Movement; molecular motors
Catalyst [Chapt 6]
enzymes responsible for all chemical reactions in organisms
© 2008 W. H. Freeman and Company
CHAPTER 3
Amino Acids, Peptides, and Proteins
아미노산, 펩티드, 단백질
Protein data bank http://www.rcsb.org/
http://www.expasy.org/
1. amino acids 아미노산
2. peptides and proteins 펩티드와 단백질
3. methods for protein study 단백질 연구
4. chemical structure of protein (= primary structure)
단백질의 화학 구조=1차구조
© 2008 W. H. Freeman and Company
function of proteins
luciferase ---producing keratin---key structural
hemoglobin -
light from luciferin and element of hair, scale,
transporting oxygen..
ATP horn, wool, nails, feathers
There is protein(s) behind almost all phenotypes
(biological phenomena: shape, function, etc)!
General structure of an amino acid .
- carboxylic acid
-Amino group
Side chain
Standard amino acids (used in translation on ribosome)
21aa (20+selenocysteine; -CH2-SeH) [cysteine, -CH2-SH]
Imino group (proline)
locating atom positions in organic compounds
Amino acids can be grouped according to R.
Full name; Glycine, Alanine…..
Three letter abbreviation
using first 3 alphabets of full name
Gly, Ala,..
exception; Ile, Trp, Gln, Asn
One letter abbreviation
use first alphabet of full name
G, A..
exception; F, W, Q, Y, N, D,
E, K, R
B (DN, Asx) Z (EQ, Glx)
Stereoisomer
-Amino acids (excepting Gly)- all optically active
L-form, and S (exception, L-Cys, R)
2. Ile, Thr has two asymmetic carbons
(2x2=4 isomers possible)
Stereoisomerism in
-amino acids .
Absorbance of the
Tyrosine and Tryptophan.
방향족 아미노산의
자외선 흡광도
Absorption of Light by Molecules
Absorption of Light by Molecules
Lambert Beer Law 람베르트-베르 법칙 log (Io/I) = cl
몰흡광계수 (1/molcm)
c, concentration of light absorbing substances 흡광시료의 농도 (mol/cm)
l, path length of light-absorbing sample, 흡광 시료의 투과길이(cm)
I/Io called transmittance (통과도) (Io , 초기 빛세기; I 투과된 빛세기)
log (Io/I) called absorbance (흡광도)
Covalent bonds between Cys stabilize further proteins
Present
in secreted proteins or
outer surface of cells
RNase, insulin
아미노산은 산과 염기로 작용할 수 있다.
Amino acid
as acid and base
Zwitterion (=dipolar ion)
meaning hybrid ion
at neutral pH
Ampholyte (=amphoteric
electrolyte)
meaning having both
acid and base in one
molecule
Amino acids could have 3 forms
Amino acids
have
Characteristic
titration curves
pKa (COOH) = 2.34
pKa (NH3) = 9.60
pI : net charge = 0
Isoelectric point or isoelectric pH (pI)
= ½ (pK1+pK2)
= ½ (2.34+9.60)
pK1, pKa of acid(+1)/conjugated base (0)
pKa of COOH
pK2 , pKa of acid(0)/conjugated base (-1)
pKa of NH3
At pI, net charge of amino acids will be
zero.
Glu, Asp
pI= ½ (pK1+pK2)
pK1,
pKa of acid(+1)/conjugated
base (0)
pKa of -COOH
pK2 , pKa of
acid(0)/conjugated base (-1)
pKa of R-COOH
His, Arg, Lys
pI= ½ (pK1+pK2)
pK1,
pKa of acid(+1)/conjugated
base (0)
pKa of R
pK2 , pKa of
acid(0)/conjugated base (-1)
pKa of NH3
Question
Why does COOH have different pKa depending
on its position?
eg)In Asp,
the pKa for -COOH is 1.88
-COOH is 3.65
Average MW of aa
=110
[average MW of
nucleotide
=330
bp= 660]
Nonstandard amino acids in protein, chemically modified after translation
비표준 아미노산- 번역후 변형PMT에 의해 생성
~300 known
Even D form of amino acid,
Nonstandard amino acids;
Reversible modification가역 변형
Nonstandard amino acids;
metabolic intermediate, neurotransmitter, poison
물질대사 중간물, 신경전달물질, 독소
2. Peptide and protein
Peptide bond connects amino acids to make a linear polymer.
Residue
잔기 Residue
Peptide bond
펩티드 결합
Linear head to tail
사슬 모양의 머리 꼬리 결합
아미노
말단
카복실 말단
Serylglycylphenylalanylalanylleucine
(ine -> yl)
SerGlyPheAlaLeu
SGFAL
(TCC) (GCT) (TTT)(GCT)(CTA)
Peptide 펩티드[dipeptide, tripeptide,… oligopeptide, polypeptide]
작은 크기
Protein 단백질-큰 크기
more than 1 polypeptide (~40~27,000 residues titin)
each polypeptide called subunit 소단위체
(protomer, 기본단위체 for homomultimeric )
monomer 단량체, dimer 이량체, trimer..oligomer protein
homomultimer, heteromulitmeric protein
Protein; more than 1 polypeptide (~40~27,000 residues)
SGFAL
How many kinds of proteins could be made out of 20 aa?
For 5 aa peptide, 20x20x20x20x20=205=3.2x106
For N long protein, 20N
for 100 long protein, 20100=1.27x10103
[atoms in universe, ~1079]
펩티드는 N 말단의 아미노기, C 말단의 카복실기
잔기의 종류에 따른 이온
생물학적 활성이 있는 펩티드와 폴리펩티드의 크기는 다양하다.
몇가지 단백질에 대한 분자 특성
단백질의
아미노산
조성
Simple proteins 단순 단백질
Conjugated protein 접합 단백질
protein (apoprotein 아포단백질)+ other chemicals (cofactor 조효소)
=holoprotein 홀로단백질
inorganic elements무기질; metals like Na, K, Mg…
organic cofactors (coenzyme 조효소 in case of enzyme);
FAD, FMN,..
**prosthetic group (tightly bound cofactor) 보조단
3. 단백질 연구
Purification of Proteins
Based on different properties of proteins
관 크로마토그라피
이온교환 크로마토그라피
크기 배제 크로마토그라피
친화 크로마토그라피
고성능 액체 크로마토그라피
High Performance liquid chromatography (HPLC)
단백질 정제 요약 표
Electrophoresis
( 전기영동)
in vaccum
field
Proportional to charge to
mass if const voltage, same
shape
In Gel,
Affected by shape, size as
well
SDS polyacrylamide gel
Electrophoresis (PAGE)
단백질의 분자량 측정
가능
등전 집중법 전기영동
Isoelectric focusing
단백질의 등전점
Two dimensional
electrophoresis
이차원 전기영동
단백질의 정량화
활성(activity)
Specific activity 단위 활성 activity per unit amount of protein
4. 단백질의 구조: 1차 구조
Structure of proteins
1o , residue sequence
2o , local regular structure
3o , 3-dimensional structure of 1 polypeptide
4o , 3-dimensional structure of multimeric protein
단백질의 기능은 아미노산 서열에 의존한다.
단백질의 1차구조가 3차원적이 구조를 결정한다.
3차원적인 입체 구조는 1차 구조에 의해 결정된다.
다형성 polymorphism
개체에 따라 기능에 큰
차이 없이 단백질
서열에 다른 차이
Determination of primary structure
1953
Frederick Sanger
Insulin
51aa,
100g, 10 years
The protein has unique sequence
(chemical structure)
Currently
A few g, a few days,
automation
직접적인 단백질 서열 결정
Amino acid Sequencing…~60aa long proteins possible
Cycles of Edman degradation 에드만 분리법 on solid phase
first removing N-terminal amino acid
& its identification by liquid chromatography
분리된 변형된 아미노산의 종류 확인-HPLC
Cleavage of disulfide bond
이황결합의 절단
폴리펩티드를 자르는 방법들
Deduction of protein aa sequence
from nucleotide sequence
Electrospray mass spectrometry (MS)
전자분무 질량분석법
MALDI MS matrix-assisted laser desorption/ionization mass
spectrometry기질 보조 레이저 탈착/이온화 질량분석법
스펙트럼은 일련의 다른 m/z 수치를 갖는 여러 피크가 나오며,
이것은 샘플 단백질의 분자량을 구하는 데 쓰인다.
Obtaining protein sequence information with tandem MS
직렬 질량분석계를 이용한 단백질 서열의 결정
Y형 이온
각 봉우리 위의 괄호안의 숫자는 아미노산 이온의 분자량
여러 개의 서열에 해당하는 y형의 분자량을 보여 준다.
Peptide
isolation from organism
genetic engineering
chemical synthesis
Application)
Study on polypeptide,
on those w/ nonstandard side chains
Or w/ isotopic labels in specific side
Development of drug
Chemical synthesis 단백질의
화학 합성
1953, Vincent du Vigneaud
oxytocin (hormone, 9aa)
1962, solid phase synthesis
C
Protection
to prevent any unwanted reaction
Protection
to prevent any unwanted reaction
Activation
chemical form for reaction to occur
Protection
to prevent any unwanted reaction
Activation
chemical form for reaction to occur
펩티드 합성시 총 수득률에 미치는 단계별 수득률의 영향
공통 서열과 서열 로고
간격을 사용하여 단백질 서열을 정렬시키기
Signature sequence 지문 서열
일정 분류군에 존재하는 특이 서열
아미노산 서열의 비교로 얻은 진화 계통도
생물의 공통 계통도
