Proline‐ and alanine‐rich N‐terminal extension of the basic bovine β‐crystallin B<sub>1</sub> chains

Berbers, Guy A. M.; Hoekman, Willeke A.; Bloemendal, H.; Jong, W.W. de; Kleinschmidt, Traute; Braunitzer, Gerhard

doi:10.1016/0014-5793(83)81013-8

Cited by 57 publications

(24 citation statements)

References 23 publications

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“…The pSl subunit is unique to the octomer [6]. pBl is resolved into two bands, pSla and pBlb, on the SDSPAGE gels [17] and the results for these were combined in Fig. 2.…”

Section: Resultsmentioning

confidence: 99%

¹H‐NMR spectroscopy of bovine lens β‐crystallin

Cooper

Carver

Truscott

1993

European Journal of Biochemistry

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'H-NMR spectroscopic studies of bovine eye lens P-crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three P-crystallin aggregates are dominated by resonances from the pB2 subunit, particularly from the N-and C-terminal extensions of this subunit. Resonances from other subunits, which all have terminal extensions, are, in general, absent from spectra of the P-crystallin aggregates. Therefore, the @2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other a-crystallin subunits. Furthermore, resonances arising from the C-terminal extension of pB2-crystallin are not present in the spectrum of the octomer, which is consistent with the C-terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C-terminal extension of pB2 and the hydrophobic pBl subunit, which is only found in the octomer, is discussed. At higher temperatures (45OC) in the octomer, partial exposure of the C-terminal extension of pS2 occurs indicating that the octomer may be starting to break up into smaller aggregates.The light refractive power of the vertebrate eye lens results from the high concentration of soluble proteins, collectively referred to as crystallins. Some 40-48% of the crystallins in the bovine lens belong to the P-crystallin family [l, 21. The seven a-crystallin subunits have molecular masses ranging over 23-33 kDa [ 3 ] . As determined by size-exclusion chromatography, the P-crystallin subunits are aggregated species consisting of dimers, trimers and octomers, which are commonly referred to as pL2, pL, and PH respectively 14-61.Based on their sequence similarity with the y-crystallins, it was proposed that each P-crystallin subunit consisted of four 'Greek key' motifs folded into two domains with the Nterminal globular domain of all P subunits having extensions of various lengths [7, 81. In addition, it was postulated that the three basic P subunits, pSl, pS2 and pB3, have extensions from the C-terminal domains [7,9]. Unlike the globular domains which are highly conserved across species, the terminal extensions of the P subunits display considerable sequence variation across species, especially within the N-terminal extension [9]. It has been proposed that the N-and Cterminal extensions extend from the globular domains [8-101. In support of this proposal, limited proteolysis of the @32 dimer results in preferential cleavage in the terminal extensions leaving the globular domains intact [ll]. The function of the terminal extensions is currently unknown, but it has been speculated that they act as attachment sites for oligomer formation or for binding to other cellular components [9].

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“…The pSl subunit is unique to the octomer [6]. pBl is resolved into two bands, pSla and pBlb, on the SDSPAGE gels [17] and the results for these were combined in Fig. 2.…”

Section: Resultsmentioning

confidence: 99%

¹H‐NMR spectroscopy of bovine lens β‐crystallin

Cooper

Carver

Truscott

1993

European Journal of Biochemistry

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“…If the fullsize, 1,593 bases, cDNA is ligated into a prokaryotic expression vector and translated in bacteria an ~60-kD protein is found on SDS-PAGE gels, which is ~10 kD larger than the calculated molecular weight (~50 kD). This increase of ~10 kD in apparent molecular weight is probably accounted for by a decreased SDS-binding capacity because of conformational properties of the alanine-proline sequences and the rigidity of the proline-rich region (Berbers et al, 1983;Vaughan et al, 1993). Posttranslational additions have to account for the other 10-kD increase of molecular weight found in eukaryotic cells.…”

Section: Characterization Of Smoothelinmentioning

confidence: 99%

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

Loop

Schaart

Timmer

et al. 1996

The Journal of Cell Biology

223

183

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Abstract. The characterization of a novel 59-kD cytoskeletal protein is described. It is exclusively observed in smooth muscle cells by Northern blotting and immunohistochemical analysis and therefore designated "smoothelin." A human smooth muscle cDNA library was screened with the monoclonal antibody R4A, and a full-size cDNA of the protein was selected. The cDNA was sequenced and appeared to contain a 1,113-bp open reading frame. Based on the cDNA sequence, the calculated molecular weight of the polypeptide was 40 kD and it was demonstrated to contain two N-glycosylation sites. Computer assisted analysis at the protein level revealed a 56-amino acid domain with homologies of ~40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, [3-spectrin and a-actinin. In situ hybridization demonstrated that human smoothelin is encoded by a single copy gene which is located on chromosome 22. Immunohistochemistry and Western blotting revealed synthesis of smoothelin in smooth muscle of species evolutionarily as far apart as human and teleost. Northern blotting indicated that sequence as well as size of the mRNA (~1,500 bases) are conserved among vertebrates. Cell fractionation studies and differential centrifugation showed that the protein cannot be extracted with Triton X-100, which indicates that it is a part of the cytoskeleton. Transfection of the human cDNA into smooth muscle cells and COS7 cells produced a protein of 59 kD, which assembled into a filamentous network. However, in rat heart-derived myoblasts association with stress fibers was most prominent. Smoothelin was not detected in primary or long term smooth muscle cell cultures. Also, transcription of smoothelin mRNA was almost instantly halted in smooth muscle tissue explants. We conclude that smoothelin is a new cytoskeletal protein that is only found in contractile smooth muscle cells and does not belong to one of the classes of structural proteins presently known.

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“…In this sense the multiple (Ala-Pro) composition of the Nterminal of the alkali light chain of myosin (above) and the N-terminal of the bovine P-crystalline B1 chains [27] can be viewed as elongated arms extending away from the rest of the molecule (cf. the collagen-like tail structures of the complement subcomponent Clq with high hydroxyproline content [28]).…”

Section: Intact Sub Fragmen T-imentioning

confidence: 99%

¹H‐NMR study of mobility and conformational constraints within the proline‐rich N‐terminal of the LC1 alkali light chain of skeletal myosin

Bhandari

Levine

Trayer

et al. 1986

European Journal of Biochemistry

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Analysis by 'H-NMR spectroscopic techniques of the conformation of the N-terminal segment of the LC1 alkali light chain of rabbit skeletal muscle has shown that this portion of the molecule adopts a well-defined elongated configuration. T h s rod-like feature is a consequence of the Ala/Pro-rich composition and the functional aspects of such conformational preference in this and similar segments in other proteins are discussed.Studies of the dynamic aspects of protein structures have demonstrated the presence of internal motions over a broad time scale (1 ps to > 1 s [l]). Specific functional roles have been ascribed to both large-scale (domain) and more localized structural fluctuations [2] as distinct from the small-scale motions that occur universally in proteins (e. g. methyl group rotations). An obvious consequence of the occurrence of such structural flexibility is that the relevant segments of the protein do not possess a unique conformation in solution.Recent NMR investigations of a variety of protein systems, e. g. the dehydrogenase multienzyme complex have identified segments of these large organized structures with mobility substantially greater than that possessed by the rest of the polypeptide(s). In the case of the pyruvate dehydrogenase complex, the segment that possesses such mobility has been identified as deriving from the region of the primary structure that links the functional domains of the lipoate acetyltransferase (E2) core [7]. The observed mobility was ascribed to facilitating the movement of the lipoyl domains between the various active sites of the enzyme complex. A characteristic of the linkage between the E2 domains is the occurrence of a comparatively high content of alanine and proline. A comparable Ala/Pro-rich sequence is found at the N-terminal 'tail' of the A1 light chain (LCI) of the myosin head [8]. This segment is observed to contribute strongly to the mobile regions identified by the 'H-NMR studies of myosin and the myosin head [9].The physical properties ascribed to these segments, namely high conformational mobility and an apparent 'random-coil' configuration, are based on the NMR spectroscopic observaCorrespondence to B. A. Levine, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, England OX 1 3 QR Abbreviation. LCI, alkali light chain of skeletal myosin; NOE, nuclear Overhauser enhancement; COSY, two-dimensional homonuclear correlated spectroscopy; SDS, sodium dodecyl sulphate; S1, skeletal myosin subfragment-I. tion of segmental flexibility of sidechain groups independent of the rotational and translational motions of the entire molecule. Substantial mobility of the backbone of these segments has been inferred. The implication of extensive (9, w ) backbone torsion angle rotations and therefore an unstructured (random-coil) configuration has no experimental verification, however, since the NMR observations could arise from coordinated motion of a peptide of up to 50 amino acid residues in length. Insight into the conformation of the polype...

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Proline‐ and alanine‐rich N‐terminal extension of the basic bovine β‐crystallin B₁ chains

Cited by 57 publications

References 23 publications

¹H‐NMR spectroscopy of bovine lens β‐crystallin

¹H‐NMR spectroscopy of bovine lens β‐crystallin

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

¹H‐NMR study of mobility and conformational constraints within the proline‐rich N‐terminal of the LC1 alkali light chain of skeletal myosin

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Proline‐ and alanine‐rich N‐terminal extension of the basic bovine β‐crystallin B1 chains

Cited by 57 publications

References 23 publications

1H‐NMR spectroscopy of bovine lens β‐crystallin

1H‐NMR spectroscopy of bovine lens β‐crystallin

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

1H‐NMR study of mobility and conformational constraints within the proline‐rich N‐terminal of the LC1 alkali light chain of skeletal myosin

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Product

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Proline‐ and alanine‐rich N‐terminal extension of the basic bovine β‐crystallin B₁ chains

¹H‐NMR spectroscopy of bovine lens β‐crystallin

¹H‐NMR spectroscopy of bovine lens β‐crystallin

¹H‐NMR study of mobility and conformational constraints within the proline‐rich N‐terminal of the LC1 alkali light chain of skeletal myosin