Laser raman spectroscopy of calf bone Gla protein

Bwchlmwa et Biophyswa Acta, 742 (1983) 525-529

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Elsevier Biomedical Press BBA 31491

LASER RAMAN SPECTROSCOPY OF CALF BONE GLA PROTEIN ACHILLE L DENDRAMIS, JAMES W POSER and EDWARD W SCHWINN

Mtamt Valley Laboratortes, Procter and Gamble Co, Cmctnnatl, OH 45247 (U S A) (Received September 28th, 1982)

Key words Raman spectroscopy, Gla protein, Conformation analysts, (Calf bone)

The Raman spectra of solid calf bone Gla protein in its native state, decarboxylated, with reduced disulfide bond, and as the calcium salt have been obtained. The amide I and III bands are consistent with the presence of a-helical, antiparallel t-sheet, and random-coil regions in all four forms of bone Gia protein. Random coil appears to be the prevailing conformation. The protein conformation in the calcium salt exhibits an increased a-helix character compared to the native protein. No significant differences in the backbone conformation are observed among the native, decarboxylated, and reduced forms of bone Gla protein. The Raman band at 504 cm -1, due to the disulfide stretching vibration in native bone Gla protein, is unchanged upon decarboxylation and binding of Ca 2÷ to the protein, indicating the absence of any changes in the conformation around the disulfide bond in these protein species. The tryptophan and most of the tyrosine residues appear to be 'exposed' rather than 'buried' in the native protein. The environment of at least one of the phenylalanine residues changes when Ca 2 ÷ is bound to bone Gla protein. A small change also appears to take place in the environment of at least one of the tyrosine residues upon Ca2+-binding or reduction of the disulfide bond.

Introduction Bone Gla protein is the most abundant noncollagenous protein in the extracellular bone matrix [1,2]. Bone Gla protein brads strongly to hydroxyapatlte, the principal mineral component of bone, and is a potent inhibitor of calcium phosphate precipitation from solutions which are supersaturated with calcium and phosphate [1,3]. The armno acid sequence of human, calf, swordfish, monkey, chicken and rat bone Gla protein have been deternuned [4-9], and the high degree of structural homology underscores the Important role this protein must play in bone metabolism. Calf bone Gla protein has a molecular weight of approx 5800 and consists of 49 amino acids, seven of which (tyrosine(4), phenylalanlne(2), tryptophan(1)) are aromatic and absorb between 250 and 300 nm. Circular dlchrolsm has been used by

Hauschka and Carr [9] to study metal ion-induced conformauonal changes in pepude backbone of chicken bone Gla protein. The same investigators have probed the enxaronment of the tyroslne and phenylalanlne residues in chicken bone Gla protern using ultraviolet difference and fluorescence spectroscopic techniques [9]. Chemical studies suggest that the location of the T-carboxyglutamic acids and the single disulfide bond, common to all species of bone Gla protein, are structural elements essential for the protein's biological function [3]. However, a role of bone Gla protein in mineral and bone metabolism has not been estabhshed. More detailed information regarding the secondary and tertiary structure of the apoproteln or when C a 2÷ is bound to It should be helpful in the understanding of its biological activity. Laser Raman spectroscopy is well suited to provide further molecular information of this na-

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ture. The work reported i n this paper was undertaken with this goal in nund. Since no spectroscopic data have been previously reported for calf bone Gla protein, the structural Information presented here is all new. Some of it is similar in nature to that presented in Ref. 9 for chicken bone Gla protein. This allows one to compare if and how the two slightly different amino acid sequences associated with the two proteins affect their secondary structures. The rest of the structural information provided is unique to Raman spectroscopy (e.g., conformation around the S-S bond) and therefore extends the present knowledge of this famdy of bone-derived proteins into aspects of secondary structure not previously addressed. Materials and Methods

Isolanon of calf bone Gla protein Bone Gla protein was isolated from calf bone by the method described by Price et al. [1]. The purified protein gave a single band on 20% polyacrylamide gel electrophoresis, had an armno acid composition consistent with that reported for bovine bone Gla protein [4], and was fully immunoreactive with antibodies specific for bovine bone Gla protein

Preparanon of bone Gla protein samples for Raman spectroscopy

multlpher tube an RCA C31034, thermoelectrlcally cooled The signal was processed by the photon-counting method. Excitation radiation at 514.5 nm was provided by a Coherent Model CR-3 argon-ion laser. The power at the sample was kept at approx 15 mW, since higher powers caused eventual sample degradaUon The protein samples, in the form of lyophihzed powders, were packed at the tip of a 2 mm outer dlamete; steel rod beanng a conical depression The spectral resolution was approx. 4.5 c m - I A grating 'filter' monochromator (Spex Lasermate) was used to reject plasma lines from the ion laser. Depending on the sample, the spectra were obtained by signal averaging 50-100 scans acqmred at a rate of 1 c m - J / s . The reproduclbihty of the spectra throughout those scans indicates that the protein is not adversely affected by our experimental conditions. Spectral frequencies are reported to + 2 cm-1. No Raman spectra could be obtained in solution due to the poor solubihty of the protein and the inherent low sensitivity of the technique Results and Discussion

Fig. 1 shows the Raman spectra of sohd calf bone Gla protein in its native state, decarboxylated, with a reduced and S-carboxarmdomethylated disulfide bond and as the calcium salt. The observed Raman bands and respective assign-

Apoproteln was prepared by dialyzing 5 mg of bone Gla protein against double-dlstdled H 2 0 The calcium salt of bone Gla protein was prepared by dialyzing 5 mg of protein against 2 mM CaC12 in double-distilled H 2 0 Following dialysis, the proteins were freeze-dried The disulfide bond in bone Gla protein was reduced and S-carboxamidomethylated using the method of Crestfield et al. [10] The y-carboxyglutamate residues in bone Gla protein were thermally decarboxylated to glutamate as described previously [3]

Laser Raman spectroscopy Raman spectra were recorded with a Spex Ramalog 5 spectrometer interfaced to an 8-bit Intel microcomputer system. The gratings were holographic, 1800 grooves/mm, and the photo-

a

" aoo le'oo'14oo 12oo looo

coo

,,- Av (cm-1)

Fig 1 Laser Raman spectra of sohd calf bone Gla protein (a) native, (b) decarboxylated (c) reduced/S-carboxamldomethylated, and (d) calcmm salt

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merits for the native protein are listed in Table I. The assignments are based on extenswe comparisons with amino a o d and peptlde spectra [11,12]. Amtde I and I I I bands In all spectra the amlde I and III band regtons are rather broad and structureless, encompassing the frequency ranges characteristic of the a-helical, antiparallel fl-sheet and random coil conformations m a polypeptide chain. The maxima positions of these bands m the native protein, 1 662 c m - 1 (amide I) and 1 260 c m - 1 (amide III), Imply that random coll is the predominant conformation [11,12]. Both of these frequency reg:ons appear unchanged m the spectrum of decarboxylated bone Gla protein When the protein is reduced, the

armde I band shifts to 1 665 cm -~, indicating a more disordered structure. The amade III region is unaffected. When calcium is bound to the protein the shaft of the amlde I band maximum from 1 662 to 1 655 cm-1 indicates an increase m a-hehcal character. The armde III reDon is less affected, but the shght bandshlft (approx. 2 c m - 1) toward higher wavenumbers is consistent with a larger a-hehcal contribution This conformatlonal change of calf bone Gla protein upon binding of Ca 2÷ is similar to that observed for chicken bone Gla protein by CD spectroscopy [8] Skeletal modes These modes involving C - - C and C - - N stretches are known to be sensitive to conforma-

TABLE I OBSERVED RAMAN LINES OF BONE GLA PROTEIN The first and tlurd columns show the wbrat~onal frequencies m wave numbers (cm- ') of the native protein sh denotes a shoulder The frequency marked with an asterisk is absent m the spectrum of the reduced protein Frequency (cm- a) 420 445 467 504 * 545 ) 578 624 644 678 720 745 760 828 ) 852 877 935 960 980 006 014 032 045 063 082 100 120 (sh)

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Tentative assignment

Frequency (cm - J) 1 161 1 175

v (S--S) Tryptophan Phenylalamne Tyrosme v (C--S) 9

Tryptophan Tyrosme Tryptophan v (C--C) Phenylalanlne Tryptophan Phenylalanme

v (C--N)

1208 1228 (sh) ] 1250 (sh) 1260 1285 (sh) 1311 (sh) "~ 1 337 f 1 363 (sh) 1450 1493 1 515 1554 ) 1585 1605 (sh) "~ 1617 5 1650 (sh) 1662 1680 (sh) 1695 (sh) 1725 ) 1752

Tentatwe assignment

Tyrosme Tyrosme and phenylalanme Anude III

CH-deformatlon, Tryptophan Tryptophan CH2-, CH3-deformaaon Hlstldme(9) Tryptophan Phenylalanme, tyrosme, tryptophan

Anude I

--COOH

528 tlon [11]. They are weak in all four spectra and remain essentially unchanged from one form of bone Gla protein to another A band at 935 cm-1 has been correlated with a-helical structure [13], although there is at least one example [14] where it is attributed to a C - - C stretching mode in the armno a o d side chains rather than the backbone. Since no slgmficant difference in the relatwe intensity of the 935 c m - 1 band is apparent between the native, reduced/S-carboxarmdomethylated and the calcmm salt of bone Gla protein, It is reasonable to assume that C - - C stretches in the amino acid side chmns are the principal contributors to the intensity of this band in bone Gla protein.

Dtsulftde bond Bone Gla protein has a single &sulfide bond [4]. The stretching vibration associated with this bond appears at 504 cm-1. This assignment is based on work with other proteins [11,12,15,16] and is supported by the absence of this band in the spectrum of the reduced and S-carboxanudomethylated protein. The S - - S stretching frequency depends on the internal rotation about the C - - S bonds [17] The observed 504 c m - 1 band suggests that the C - - C - - S - - S - - C - - C linkage may be best described by a gauche-gauche-gauche pattern. Since the frequency remains unchanged when Ca 2÷ is bound to the protein, the lmphcatlon is that the increased a-helix character of the backbone conformation does not affect the conformation around the disulfide bond The C - - S stretch, which is sensitive to the conformation of the C - - C bond adjacent to the S - - S bond [18], is too weak to be observed clearly in our spectra The weakness of this mode is not unusual and has been linked with large C - - S - - S bond angles (larger than 110 °) [19]. A broad, weak, featureless band at 678 cm-~ in the native protein spectrum may be due to the C - - S stretch, although its absence in the spectra of the decarboxylated and calcium salt of bone Gla protein makes this assignment questionable

SMe-cham residues The majority of the observed Raman bands are attributable to aromatic side chains (see Table I). Calf bone Gla protein has four tyrosine, one tryptophan and two phenylalanine residues. The 852

and 828 c m - t bands of tyrosine result from Fermi resonance between a symmetric ring-breathing and the overtone of an out-of-plane v~bratIon of the para-substituted benzene ring [20]. The relative intensities of these bands are related to the environments of the tyroslne side chains [21]. The intensity ratio of the 852 and 828 c m - l tyroslne bands in the native protein (I852/I828 = 10/8) indicates that most of the tyroslne residues are normally exposed rather than 'buried'. This ratio remains unchanged in the spectrum of the decarboxylated protein, while it Increases shghtly (10/7) in the spectra of reduced and S-carboxamldomethylated and the calcium salt of bone Gla protein This suggests that at least one of the four tyrosine residues in the native protein becomes more exposed upon reduction of the disulfide linkage or binding of Ca 2+ The lack of a sharp R a m a n line at 1361 cm-1 suggests that the single tryptophan residue may also be exposed [22,23]. Tins is supported by the low intensity of the 877 cm -1 tryptophan band relative to that of the conformationally nonsensltlve CH~ bending vibration at 1450 cm -1 (I88o/I145o = 0.1) [24]. Since the tryptophan band at 877 c m - • appears unchanged in the spectra of all bone Gla protein species, we have to assume that the environment of the tryptophan residue does not change appreciably in any of the modified forms of the native protein In contrast, the intensity of the phenylalanlne ring breathing vibration at 1006 cm -~ changes significantly when Ca 2+ is bound to the protein This indicates that the environment of at least one of the two phenylalanine residues changes as the Ca 2+-bmdlng takes place From a comparison of our solid state data with the solution data of Ref. 9, it appears that, despite some differences in the primary structure between calf and chicken bone Gla protein, there are many similarities in their secondary structure These are (1) the prevadmg random coil conformation in the native state, (2) the basically unchanged secondary structure upon decarboxylatlon, and (3) the marked increase In a-helix character when Ca 2+ is bound to the protein. In addition, both studies suggest that binding of Ca 2+ affects the environments of some of the phenylalanine and tyrosine residues The apparent discrepancy regarding the degree of

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exposure of the tyroslne residues for the p r o t e i n calcium salt ( b i n d i n g of Ca 2+ to chicken b o n e G l a p r o t e i n induces decreased exposure of tyrosme residues [8]) m a y be due to the k n o w n presence of a n a d d i t i o n a l tyrosine residue in calf b o n e G l a protein. Since the c o n f o r m a t i o n of a protein in solution m a y be different t h a n that in the solid state (whether as a crystal or lyoplulIzed powder), conclusions d r a w n from such comparisons should always be kept in perspective. The present knowledge of the structure of b o n e G l a p r o t e i n a n d its b i n d i n g properties m a y be greatly e n h a n c e d b y using ultraviolet-exoted reson a n c e R a m a n spectroscopy to study b o n e G l a p r o t e i n in solution or In the sohd state b o u n d to a hydroxyapatite surface. I n addition, several of the ions m the l a n t h a n l d e series are k n o w n to b i n d to b o n e G l a protein [9] a n d can induce conformational changes sinular to that caused b y Ca 2+. Thus, one m a y use laser-induced fluorescence techniques [25] to learn more a b o u t the specific m e t a l - i o n - b i n d i n g sites in b o n e G l a protein a n d the relative position of these s~tes with respect to the aromatic a m i n o acid residues. Both types of spectroscopic studies are currently u n d e r w a y in o u r laboratory.

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