Studying the structure of proteins and the factors involved

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Proteins are macromolecules that serve as the functional terminal of the Central Dogma of Molecular Biology (Crick, 1970). The biological function of each protein is intricately related to its structure. The structure of the protein by itself is very fragile and in turn, depends on the environment it is in, for its stabilization (Pauling L et al, 1951). Hence apart from the study of protein structure itself, various studies have been carried out over the factors that affect protein structure (Burder D, 2007) because knowledge about these factors can not only contribute to better administration and preservation of therapeutic proteins, but also provide an insight into diseases that are caused by the structural alterations of proteins (Dobson CM, 1999; Dennis S, 2003; Chiti F and Dobson CM, 2006).

When studies have been carried out and proved beyond doubt the dependence of protein structure on ambient pH (Heremans L and Heremans K, 1989; Boye J et al, 2009) and temperature (Tilton RF et al, 1992), as well as the presence of certain chemicals like cross-linking agents, reducing agents, alkylating agents, halogenating agents, etc… (Anfinsen C, 1961; Hirschmann R et al, 1969; Gutte B and Merrifield R, 1971), very few studies have been carried out about the effect of deforming mechanical stress and shear upon the structure of proteins.

Proteins are made of amino acids each of which has a particular isoelectric pH at which it exists in a zwitterionic form, which again depends on the nature of the side chains of the amino acids. So also, a protein's isoelectric pH is that pH at which the side chains of the various amino acid residues are ionized. This ionization is necessary for biological function of proteins such as when the residues form the catalytic groups of an enzyme whose ionized states alone can help with the mechanism of catalytic reaction (Harris TK and Turner GJ, 2002; Benkovic et al, 2003). Apart from that, for the formation of electrostatic bridges between various parts of the same protein or between the various subunits of the same protein that hold the protein structure together, the maintenance of certain polar amino acids in the ionized state at a particular narrow range of pH to facilitate this interaction is very important (Marqusee S and Sauer RT, 1994; Xu D et al, 1997). Hence pH affects protein structure and its function.

Higher ordered structures of proteins such as secondary (α helices and β sheets), super-secondary (motifs and domains) and tertiary structures are stabilized by often non-covalent weak interactions. In fact, with the exception of the disulphide bond, all the other molecular interactions in the protein's higher order structures are all non-covalent weak bonds (Chiang YS et al, 2007). These include:

Hydrogen bond

Hydrophobic interaction (Van der waal's forces)

Electrostatic interactions

Being weak bonds, they are all sensitive to higher temperature which alters the structure and renders the protein biologically inactive (denatured) either temporarily or permanently, thus making the proteins structure temperature dependent.

Reducing, alkylating, halogenating, cross-linking agents, etc… also disrupt one or more of the above interactions and also the covalent forces like disulphide bonds in the protein thus altering its structure (Anfinsen C, 1961; Hirschmann R et al, 1969; Gutte B and Merrifield R, 1971) and so the protein structure is dependent on the these chemicals.

Thus in short, polypeptide chains are synthesized in a particular physiological environment and folded in a similar conducive environment into a biologically functional protein and the protein is more or less stable only in that environment. When it is taken outside that environment either for in vitro studies, pharmacological purposes or because it has its function elsewhere, it is not so stable. All proteins are synthesized within the cytoplasm of a cell in a colloidal aqueous environment that is devoid of any rigorous movement that imparts a great shear force on these proteins. In unicellular organisms, even if they are motile and use tubulin, actin and kinesin fibres of the cytoskeleton for cytoplasmic streaming and propulsion, there is hardly any shear stress on these proteins.

But the scenario is grossly different for multicellular organisms with complex differentiated tissue and division of labor. In tissue responsible for locomotion, circulation and structural tissue, shear is obvious. A few simple instances are the repeated shear on the contractile muscle proteins, especially involuntary muscles like those of GI tract and heart that never stop their movement, rubbing of the synovial membrane proteins with the synovial fluid proteins in the skeletal muscle joints and the shear on the proteins that circulate in blood due to their friction with the viscous blood plasma and the walls of the capillaries and blood vessels.

In these situations, proteins will be experiencing large shear forces which could cause a possible relative displacement of the various secondary structures, domains and motifs with respect to one another and thus affect the protein function (Jaspen and Hagen, 2006). Also important are situations such as plasma transfusion, blood screening, the cardiopulmonary bypass pump (heart-lung machine), etc… where the proteins in blood experience large shearing stress.

The intravenous administration of proteins of therapeutic value such as surface antigens in vaccination, immunoglobulins, plasma proteins, glycoprotein and peptide hormones, etc… via the thin inner bore of the needle of a syringe also confer upon these proteins enormous shear force within a short duration especially with the pressure and velocity of the protein solution in the syringe being much higher than what it is physiologically subjected to.

Here, for immunoglobulins and surface antigens, it is very important that they maintain their structure as such because a change in the antigen structure from what the pathogen has will make the body synthesize antibodies complementary in structure to the altered antigen and hence provide immunity against the structurally anomalous antigen instead of the antigen with the correct structure found in nature against which we seek protection. So also alteration in the structure of the injected antibody may make it either incapable of binding to the antigen we want to opsonize and eliminate, or alter its affinity by making it have a structure complementary to that of a self-antigen and thus trigger a highly dangerous auto-immune response with far-reaching and sometimes fatal consequences. The fact that auto-immune diseases crop up suddenly in healthy individuals with a clean medical history on some random day, usually after they are about 35-40 years of age, casts some suspicion on whether the auto-antibodies are generated accidentally by mechanical shear of normal antibodies despite the body's elaborate negative clonal selection of auto-reactive immune cells.

So in this study, we aim to experimentally check whether proteins undergo any structural alteration when the solutions are subjected to mechanical shear in intravenous syringe needles of various inner diameters at various velocities, to detect and measure the change in structure of the protein by monitoring its absorption spectra using a Fourier Transform Infrared (FT-IR) spectrophotometer and calculate the shear experienced by the protein.

If any structural changes occur in the protein, the structural shift cause changes in the vibrational freedom of the various atoms in the protein molecule consequently altering its resonant frequency, as well as the pattern of Raman (Stoke's and anit-Stoke's) scattering. This alters the absorbance of IR of a particular wavelength out of the set of wavelengths supplied by the FT-IR spectrophotometer.

A protein is normally made of α helices and β sheets together with some turns, loops and random coils. Each of these structures absorbs IR radiation at its own narrow range of wavelength (or wave number). So the content of the secondary structure components in the normal protein may be known. During mechanical shear, the most common alteration in structure will be the randomization of α helices and β sheets. It will be denoted by a sharp decrease in the absorbance at the peaks of the spectrum regions which will be absorbed by α helices and β sheets and will show a sharp increase in those wave numbers of the spectrum that will be absorbed by random coils. Hence by peak-fitting the absorption spectra of the normal protein with that obtained after the protein has been subjected to mechanical shear, if there are any changes in the absorption spectra, it proves the protein structure has been altered by mechanical shear in the syringe needle and an analysis of the peaks give an idea of the secondary structures that have been affected the most by mechanical shear of passing through the narrow bore of the syringe needle.

However, the interpretation of structure from the FT-IR absorption spectrum is not so very straightforward. It requires that we assign the various absorption peaks to the various bands that are formed because the peptide group, the structural repeat unit of proteins, gives up to 9 characteristic bands named amide A, B, I, II ... VII. The amide A band (about 3500 cm-1) and amide B (about 3100 cm-1) originate from a Fermi resonance between the first overtone of amide II and the N-H stretching vibration. Amide I and amide II bands are two major bands of the protein infrared spectrum. The amide I band (between 1600 and 1700 cm-1) is mainly associated with the C=O stretching vibration(70-85%)and is directly related to the backbone conformation. Amide II results from the N-H bending vibration (40-60%) and from the C-N stretching vibration (18-40%). This band is conformational sensitive. Amide III and IV are very complex bands resulting from a mixture of several coordinate displacements. The out-of-plane motions are found in amide V, VI and VIII.

protein_spec.gif

Amide A is with more than 95% due to the the N-H stretching vibration. This mode of vibration is not depend on the backbone conformation but is very sensitive to the strength of a hydrogen bond (between 3225 and 3280 cm-1 for hydrogen bond length from 2.69 to 2.85 angstrom, (Krimm & Bandekar Adv Protein Chem 1986;38:181-364).

Amide I is the most intense absorption band in proteins. It is primilary goverend by the stretching vibration of the C=O (70-85%) and C-N groups (10-20%). Its frequency is found in the range between 1600 and 1700 cm-1. The exact band position is determined by the backbone conformation and the hydrogen bonding pattern.

Amide II is found in the 1510 and 1580 cm-1 region and it is more complex than amide I. Amide II derives mainly from in-plane N-H bending (40-60% of the potential energy). The rest of the potential energy arises from the C-N (18-40%) and the C-C (about 10%) stretching vibrations.

Amide III, IV are very complex bands dependent on the details of the force field, the nature of side chains and hydrogen bonding. Therefore these bands are of little use.

Amino acid side chain vibrations

The presence of bands arising from amino acid side chains must be recognized before attempting to extract structural information from the shapes of amide I and amide II bands. The contribution of the side chain vibrations in the region between 1800 and 1400 cm-1 (amide I and amide II region) has been thoroughly investigated by Venyaminov & Kalnin 1990 (Biopolymers 1990;30(13-14):1243-57). Among the 20 proteinogenous amino acids only 9 (Asp, Asn, Glu, Gln, Lys, Arg, Tyr, Phe, His) show a significant absorbance in the region discussed above. The contribution of the different amino acid side chains were fitted by a sum of Gaussian and Lorentzian components.

AS

vibration

cm-1

A0

(l/mol/cm)

FWHH

(cm-1)

surface

(x10-4 l/mol/cm)

Asp

-COO st as

pH>pK (~4.5)

1574

380

44

5.5

-COOH st

pH<pK (~4.5)

1716

280

50

4.1

Glu

-COO st as

pH>pK (~4.4)

1560

470

48

7.1

-COOH st

pH<pK (~4.4)

1712

220

56

3.6

Arg

-CN3H5+ st as

1673

420

40

4.3

st s

1633

300

40

3.6

Lys

-NH3+ bd as

1629

130

46

1.8

bd s

1526

100

48

1.3

Asn

-C=O st

1678

310

32

2.7

-NH2 bd

1622

160

44

2.5

Gln

-C=O st

1670

360

32

3.1

-NH2 bd

1610

220

44

3.5

Tyr

ring-OH

pH<pK (~10)

1518

430

8

1.0

ring-O

pH>pK (~10)

1602

160

14

0.7

1498

700

10

2.5

His

ring

1596

70

14

0.3

Phe

ring

1494

80

6

0.2

terminal

-COO st as

1598

240

47

3.5

-COOH st

1740

170

50

2.1

-NH3+ bd as

1631

210

54

3.8

bd s

1515

200

60

4.3

-NH2 bd

1560

450

46

7.5

frequency, absorbance at the maximum (Ao), full width at half height (FWHH), surface of Gaussian band

st=stretching vibration

bd=bending

s=symetrical

as=asymetrical

(according to Venyaminov & Kalnin Biopolymers 1990;30(13-14):1243-57)

a)

b)

sec. structure

Mean (cm-1)

RMS (cm-1)

Max (cm-1)

Mean (cm-1)

RMS (cm-1)

Max (cm-1)

Region (cm-1)

turns

1694

1.7

2

-

-

-

1688

1.1

2

-

-

-

1683

1.5

2

1678

2.1

5

1682-1662

1670

1.4

2

1670

2.9

5

1663

2.2

4

1664

1.0

3

alpha-helix

1654

1.5

3

1656

1.5

3

1648

1.6

3

1662-1645

unordered

1645

1.6

4

1641

2.0

3

1645-1637

beta sheet

1624

2.4

4

1624

2.5

5

1631

2.5

3

1633

2.1

4

1637-1613

1637

1.4

3

-

-

-

1675

2.6

4

1685

2.1

4

1689-1682

Proteins in solution (Susi & Byler (Methods Enzymol 1986;130:290-311), a) as hydrated film on a ATR plate (Goormaghtigh et al. Eur J Biochem 1990 Oct 24;193(2):409-20, b) the mean frequency of each component is reported with the root mean square (RMS) and the maximum deviation (Max).

Thus once we take all these factors into account, the shape of the amide I band of globular proteins is characteristic of their secondary structure. With a publication by Byler & Susi (Biopolymers 1986 Mar;25(3):469-87) the determination of secondary structures in proteins from FTIR spectra really started. This was possible by the availability of high signal-to-noise ratio digitalized spectra obtained by the FTIR spectrometer and by the access to computers and software able to perform many operations on the spectra in a short time.

With the use of FT-IR spectrophotometer, protein structural characterization in diverse environments is possible. In many cases it is not sufficient to just have the three-dimensional structure of a protein in aqueous or in the crystalline state. Often information on the structural properties of a protein is required in conditions that resemble the actual environment of the protein in vivo such as in the presence of phospholipid membranes (for membrane proteins), organic buffers, detergent micelles, and so on. Fourier transform infrared spectroscopy (FTIR) is one of the few techniques that can be applied for structural characterization of proteins in such environments to get dynamic in vivo observations of the protein. Also with the advent of Attenuated Total Reflectance (ATR) technology and the use of the ATR plate in FT-IR makes it possible to use protein samples as in without any preparation irrespective of whether it is a solid or solution. In Total Internal Reflection (TIR), there is no loss in intensity of light even if it undergoes multiple TIRs and thus it avoids the problem of strong attenuation of the IR signal in highly absorbing media, such as aqueous solutions.

Materials

Instruments

Cold room maintained at 2°C under low light conditions

Homogenizer/blender

Refrigerator

Sonicator

pH meter

Lyophilizer

Autoclave

Refrigerated centrifuge capable of 40,000 g (7,500 rpm)

Analytical ultracentrifuge like SW41-Ti capable of 288,000 g (41,000 rpm)

Spectrophotometer

Spectrofluorimeter

Fourier Transform Infrared Spectrophotometer (FT-IR)

Gel slabs and equipments for electrophoresis

Cryo X-ray diffractometer capable of 12 A° resolution

Clark oxygen electrode or calomel electrode

Electronic balance capable of microgram measurement

Glassware such as standard flasks, test tubes, glass rod, beaker, etc…

Reagents

S.No

Reagent

Purpose

1

Millipore grade water

Solvent

2

Liquid nitrogen

Cryopreservant

3

Maltose

Gradient

4

Diethyl pyrrocarbonate (DEPC)

Water treatment

5

Potassium hydroxide (KOH)

pH adjustment

6

Sodium chloride (NaCl)

Maintain osmolarity

7

Magnesium chloride (MgCl2)

8

Sorbitol

Cryoprotectant

9

Ethylene diamine tetra acetic acid (EDTA)

Chelating agent

10

Benzamidine hydrochloride

Protease inhibitors

11

ε-Aminocaproic acid

12

Tricine

Good's buffers

13

Hydroxy ethyl piperizine ethane sulfonic acid (HEPES)

14

Triton X 100

Detergents

15

N-Dodecyl-α-D-maltopyranoside (α-DM)

Table 2: List of reagents required for PSII isolation.

Solutions

Methods

Results

Discussion

Conclusion

Appendices

References and citation

Crick F (August 1970) "Central dogma of molecular biology" Nature 227 (5258)

Pauling L, Corey RB, Branson HR (1951) "The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain" Proc Natl Acad Sci USA 37 (4): 205-211

David W Burden (2007) Factors Affecting Protein Stability In Vivo, Quart Rev of Biophysics Issue No. 4, June 9, 2007

Dobson CM (1999), "Protein misfolding, evolution and disease", TIBS 24 (9): 329-332. PMID 10470028

Dennis J Selkoe (2003), "Folding proteins in fatal ways", Nature 426 (6968): 900-904. PMID 14685251

Chiti, F and Dobson, C (2006) "Protein misfolding, functional amyloid, and human disease" Annual review of biochemistry 75: 333-366. PMID 16756495

Luc Heremans and Karel Heremans (1989) "Raman spectroscopic study of the changes in secondary structure of chymotrypsin: effect of pH" Biochimica et Biophysica Acta Volume 999, Issue 2, 30 November 1989, Pages 192-197

Joyce I. Boye, Ashraf A. Ismail and Inteaz Alli (1996) "Effects of physicochemical factors on the secondary structure of β-lactoglobulin" Journal of Dairy Research 1996, 63: 97-109

Robert F Tilton Jr, John C Dewan, Gregory A Petsko (1992) "Effects of temperature on protein structure and dynamics: x-ray crystallographic studies of the protein ribonuclease A at nine different temperatures from 98 to 320K" Biochemistry, 1992, 31 (9), pp 2469-2481

Anfinsen CB and White FH (1961) "Kinetics of Protein Stability and effect of reducing agents" J Biol Chem 236, 1353, 1961

Hirschmann R, Nutt RF, Veber DF, Vitali RA, Varga SL, Jacob TA, Holly FW, Denkewalter G (1969) "Chemicals that affect preparation of enzymatically active material" J Amer Chem Soc, 91, 507, 1969

Gutte B and Merrifield RB (1971) "Chemicals affecting stability of protein secondary structure" J Biol Chem 246, 1922, 1971

Thomas K Harris and George J Turner (2002) "Structural Basis of Perturbed pKa Values of Catalytic Groups in Enzyme Active Sites" IUBMB Life, 53: 85-98, 2002

Stephen J Benkovic and Sharon Hammes-Schiffer (2003) "A Perspective on Enzyme Catalysis" Science, August 2003 Vol 301 no 5637 pp 1196-1202

Susan Marqusee, Robert T. Sauer (1994) "Contributions of salt bridge network to the stability of protein secondary structure" Protein Science Volume 3, Issue 12, pages 2217-2225, December 1994

Xu D, Tsai CJ and Nussinov R (1997) Protein Stability and Electrostatic Interactions" 1997 JMB 265, 68 - 84

Chiang YS, Gelfand TI, Kister AE, Gelfand IM (2007) "Review of supersecondary structures of proteins" Proteins 68 (4): 915-921

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