TÉCNICAS E INSTRUMENTOS DE RECOLECCIÓN DE DATOS

Differences in the spectra of fragment D (Mw 100 KDa) and fragment D* (Mw 90 KDa) are expected to reflect the structural features absent in fragment D* and present in the G-chain between residues 303-411 in the original fragment D. The percentages of secondary structures in fragment D* are similar to those of fragment D, indicating that the removal of the polypeptide G-chain (Mw lOKDa) does not result in significant changes in the secondary structure. The slight differences observed in the comparison of the two spectra are not so marked as to suggest an indication of the pre­ dominant secondary structure of the missing structures of the G-chain (Shimizu et al., 1992). The high rate of amide H/^H exchange suggests that fragment D* has a less compact structure than that of fragment D, possibly caused by the removal of the G-chain segment.

3.5. SUMMARY.

The secondary structure of human fibrinogen and its plasmin-fragments have been studied by FTIR spectroscopy. The percentages of secondary structure in fibrinogen are 37% a-helix, 3 0% 6 -sheet and 14% turns, in good agreement with

CD spectroscopic studies. The spectra of fibrin clots formed in Ca^'*’-containing buffer have a lower amide I/amide II ratio than fibrin clots in the presence of the quelator EGTA, which is interpreted as being due to aggregation.

Plasmin-digested fragments of fibrinogen were isolated and studied. Fragment E (Mw 45 KDa) contains 50% a-helix, attributed to its solvent-exposed coiled-coil domain, with m inor cont r i b u t i o n s of 6 -strands and turn structures.

Fragment D (Mw 100 KDa) shows 35% a-helix, 29% 6 -sheet and

17% turn secondary structures. Of the two domains present in Fragment D, the coiled-coil portion (Mw 27 KDa) was isolated and shows a percentage of 70% in a-helical structures. The thermolabile globular domain is estimated to be twice richer in 6 -sheet structures than a-helical structures.

The secondary structure of Fragment D* (Mw 90 KDa) has been shown to be similar to Fragment D. Fragment D* shows a solvent-accessible structure as indicated by the rapid rate of H/^H exchange.

CHAPTER 4.

BIOPHYSICAL STUDIES OF THE PFl AND FD COAT PROTEINS IN THE PHAGE, IN DETERGENT MICELLES AND IN PHOSPHOLIPID MEMBRANES.

4.1.INTRODUCTION.

Filamentous phages are the thinnest and longest viruses known, consisting of a single-stranded DNA encapsulated by 2000-7000 copies of a 5 KDa coat protein in a characteristic filament p a r t i c l e (Marvin & Wachtel, 1975; Day et al., 1988) . Depending on the strain, the viruses measure 6 nm in

diameter by 1 0 0 0 - 2 0 0 0 nm long, and contain about 6 -1 0 % (w/w)

DNA and no lipid.

During the life cycle of the phages the coat proteins have to adapt to two different environments, the bacterial membrane and the phage. During the synthesis of the viral components inside the bacteria, the coat proteins are enco­ ded as membrane proteins with a signal seguence (Smilowitz et al., 1972; Webster & Cashman, 1973) which is removed by the host signal-peptidase. Phage assembly seems to take place at points of the inner bacterial membrane where the coat proteins and the DNA cluster as the filament particles are extruded out to the external media.

On the basis of the structure of the viruses and the homology of the aminoacid sequence of the coat proteins, two classes of filamentous phages can be distinguished: class I that correspond to the fd, M13, fl strains and class II to the Pfl and Pf 3 strains (Day et a l . , 1988). The aminoacid sequence of the coat proteins, such as those of the Pfl and fd phages (table 4.1), shows a pattern of an acidic N-termi-

TABLE 4.1 .

Aminoacid sequence of the fd coat protein.

Ala-Glu-Gly-Asp-Pro-Ala-Lys-Ala-Ala-Phe^Q-Asp-Ser-Leu-Gln-Ala Ser-Ala-Thr-Glu-TyrgQ-Ile-Gly-Tyr-Ala-Trp-Ala-Met-Val-Val-IlegQ Val-Gly-Ala-Thr-Ile-Gly -Ile-Lys-Leu-Phe^Q-Lys-Lys-Phe-Thr-Ser Lys-Ala-Ser^g

Aminoacid sequence of the Pfl coat protein.

Gly-Val-Ile-Asp-Thr-Ser-Ala-Val-Glu-SeriQ-Ala-Ile-Thr-Asp-Gly Gln-Gly-Asp-Met-LySgQ-Ala-Ile-Gly-Gly-Tyr-Ile-Val-Gly-Ala-LeUgQ Val-Ile-Leu-Ala-Val-Ala-Gly-Leu-Ile-Tyr^Q-Ser-Met-Leu-Arg-Lys Ala4g

nal segment, a basic C-terminus and a 20-residue hydrophobic region in the centre of the sequence.

Neutron and X-ray diffraction studies of oriented phages (Marvin & Wachtel, 1975; Day et al., 1988) indicate that the coat proteins exist as a-helical subunits parallel to the phage axis, with the solvent-exposed N-termini covering the hydrophobic region of adjacent coat proteins in a array mimicking scales in fish. The arrangement of the a-helical subunits forms left-handed spirals around the filamentous particle, with 4.5-5.5 spirals per turn (Day et al., 1988).

In the bacterial membrane, the hydrophobic segment is thought to span the bilayer with the C-termini facing the cytoplasmic side and the N-terminus the periplasmic side (Wickner, 1975). CD spectroscopic studies indicate that the a-helical content of the Pfl and the fd coat proteins is reduced markedly upon solubilisation in detergent micelles ? or reconstitution in lipid aqueous suspensions as compared ^ to the phage (Schiksnis et al., 1987; Leo et a l . , 1987). The tertiary structure of the Pfl coat protein in DPC detergent micelles has been determined using 2-D ^^N/^H-NMR spectros­ copy. The Pfl coat protein, in this detergent micelle, is a monomer and adopts an arrangement of two stable a-helical segments (Shon et al., 1991; Schiksnis at al., 1987). With the assumption that this structure was conserved in lipid membranes, solid-state NMR spectroscopy of ^H/^^N-labelled

residues of the Pfl protein was used to deduce the degree of motion and the orientation of the two helical stretches adopted by the protein in the membrane. These studies led to Shon and coworkers (1991) to propose a model for the Pfl protein in the phospholipid membrane where the hydrophobic region (about 24 residues) forms a membrane-spanning a-helix whilst the amino-terminal region (residues 1-14) is arranged as an amphipathic a-helix lying parallel to the lipid bila­ yer plane.

The c o n f o r m a t i o n of the M13 coat p r o t e i n has been studied in SDS micelles by NMR spectroscopy (Henry & Sykes, 1992). The sequence of the M13 protein is identical to the fd protein, except for a replacement of Aspll to Asn (Day et al., 1988). The M13 coat protein in the micelle exists in a si m i l a r c o n f o r m a t i o n to that of the Pfl p r o t e i n in DPC micelles (Henry & Sykes, 1992). In the micelle, the M13 coat protein forms a dimer with both hydrophobic regions juxta­ posed in a parallel fashion. Solid-state ^H/^^N-NMR studies of the fd protein in the lipid membrane indicate a conforma­ tion that is rigid between residues Ala6-Phe41 and mobile in both N- and C-termini (Leo et al., 1987).

The coat protein of the filamentous phages, especially the fd and Pfl proteins, are used extensively as models for the d e t e r m i n a t i o n of the s t r u cture and the d y n a m i c s of

membrane proteins in membrane-mimicking environments such as ^ detergent micelles and phospholipid aqueous systems (Schik-

^ snis et al., 1987; Gallusser & Kuhn, 1990). Owing to their L small m o l e c u l a r weight, it is p o s s i b l e to s tudy basic features of the structure and dynamics of the exoplasmic and the membrane-spanning domains of membrane proteins. Herein, FT-IR spectroscopy is used to study the conformation of the Pfl and fd coat proteins in the phage, in detergent micelles and reconstituted in a lipid membrane environment. Internal reflectance FT-IR spectroscopy is used to study the orienta­ tion of the secondary structure of the two coat proteins within the lipid bilayer. Other biophysical techniques such as polarization of the fluorescent probe DPR and calorimetry are used to examine the degree of lipid chain perturbation caused by the Pfl coat protein when embedded within the lipid bilayer.