Reversible unfolding of the gelatin-binding domain of fibronectin: structural stability in relation to function.


Abstract

Fibronectin, a large multidomain glycoprotein, binds denatured collagen (gelatin) and mediates cell attachment and spreading on collagen-coated surfaces. Despite the high affinity, binding to gelatin is disrupted by relatively mild conditions. We have examined the effects of denaturants on the structure and function of a 42-kDa gelatin-binding fragment (GBF) isolated from chymotryptic and thermolytic digests of the parent protein. Application of linear gradients to GBF-loaded gelatin-agarose columns resulted in peak elution of the fragment at pH 5.2 or 10.2, at 0.4 M dimethylformamide, 0.9 M GdmCl, or 2.0 M urea, conditions far short of those required to induce structural changes detectable by fluorescence or circular dichroism. Solvent perturbation, fluorescence quenching, and chemical modification experiments indicate that about half of the 8 tryptophans, one-third of the 21 tyrosines, and all of the 9 lysine residues are solvent-exposed in the native protein and that 1 or more of the latter are directly involved in binding to gelatin, most likely through a hydrogen-bonding mechanism. Titration with GdmCl produced a single unfolding transition centered near 2.5 M GdmCl as monitored by changes in fluorescence and circular dichroism. This transition was fully reversible with complete recovery of structural parameters and gelatin binding. Treatment with disulfide reducing agents caused rapid irreversible changes in structure similar to those produced by GdmCl with concomitant loss of gelatin binding. Thus, tertiary and secondary structures are important for binding, but binding can be disrupted without perturbing those structures. Study holds ProTherm entries: 3837 Extra Details: NaN3(0.02%) was added in the experiment gelatin-binding fragment; structural changes;,hydrogen-bonding mechanism; tertiary and secondary structures

Submission Details

ID: qAqTAG2E

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:22 p.m.

Version: 1

Publication Details
Isaacs BS;Brew SA;Ingham KC,Biochemistry (1989) Reversible unfolding of the gelatin-binding domain of fibronectin: structural stability in relation to function. PMID:2713351
Additional Information

Number of data points 1
Proteins Fibronectin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dG_H2O
Libraries Mutations for sequence MLGGPGPGLLLLLAVLSLGTAVPSAGASKSRRQAQQIVQPQSPLTVSQSKPGCYDNGKHYQINQQWERTYLGSALVCTCYGGSRGFNCESKPEPEETCFDKYTGNTYRVGDTYERPKDSMIWDCTCIGAGRGRISCTIANRCHEGGQSYKIGDTWRRPHETGGYMLECVCLGNGKGEWTCKPIAEKCFDQAAGTSYVVGETWEKPYQGWMMVDCTCLGEGSGRITCTSRNRCNDQDTRTSYRIGDTWSKKDNRGNLLQCICTGNGRGEWKCERHTSLQTTSAGSGSFTDVRTAIYQPQPHPQPPPYGHCVTDSGVVYSVGMQWLKTQGNKQMLCTCLGNGVSCQETAVTQTYGGNSNGEPCVLPFTYNGKTFYSCTTEGRQDGHLWCSTTSNYEQDQKYSFCTDHTVLVQTRGGNSNGALCHFPFLYNNHNYTDCTSEGRRDNMKWCGTTQNYDADQKFGFCPMAAHEEICTTNEGVMYRIGDQWDKQHDMGHMMRCTCVGNGRGEWTCVAYSQLRDQCIVDGITYNVNDTFHKRHEEGHMLNCTCFGQGRGRWKCDPVDQCQDSETRTFYQIGDSWEKYLQGVRYQCYCYGRGIGEWACQPLQTYPDTSGPVQVIITETPSQPNSHPIQWSAPESSHISKYILRWKPKNSPDRWKEATIPGHLNSYTIKGLRPGVVYEGQLISVQHYGQREVTRFDFTTTSTSPAVTSNTVTGETTPLSPVVATSESVTEITASSFVVSWVSASDTVSGFRVEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRKYTVNVYEISEEGEQNLILSTSQTTAPDAPPDPTVDQVDDTSIVVRWSRPRAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGVQYNITIYAVEENQESTPVFIQQETTGVPRSDKVPPPRDLQFVEVTDVKITIMWTPPESPVTGYRVDVIPVNLPGEHGQRLPVSRNTFAEVTGLSPGVTYHFKVFAVNQGRESKPLTAQQATKLDAPTNLQFINETDTTVIVTWTPPRARIVGYRLTVGLTRGGQPKQYNVGPAASQYPLRNLQPGSEYAVSLVAVKGNQQSPRVTGVFTTLQPLGSIPHYNTEVTETTIVITWTPAPRIGFKLGVRPSQGGEAPREVTSESGSIVVSGLTPGVEYVYTISVLRDGQERDAPIVKKVVTPLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGYSLEEVVHADQSSCTFENLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNVGPDTMRVTWAPPSSIELTNLLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYLVSVSSVYEQHESIPLRGRQKTALDSPSGIDFSDITANSFTVHWIAPRATITGYRIRHHPENMGGRPREDRVPPSRNSITLTNLNPGTEYVVSIVALNSKEESLPLVGQQSTVSDVPRDLEVIAATPTSLLISWDAPAVTVRYYRITYGETGGSSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPVSINYRTEIDKPSQMQVTDVQDNSISVRWLPSSSPVTGYRVTTAPKNGPGPSKTKTVGPDQTEMTIEGLQPTVEYVVSVYAQNQNGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEETAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTTIPAPTNLKFTQVTPTSLTAQWTAPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAIPANGQTPIQRTIRPDVRSYTITGLQPGTDYKIHLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIQVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVPSTVQKTPFITNPGYDTGNGIQLPGTSGQQPSLGQQMIFEEHGFRRTTPPTTATPVRHRPRPYPPNVNEEIQIGHVPRGDVDHHLYPHVVGLNPNASTGQEALSQTTISWTPFQESSEYIISCHPVGIDEEPLQFRVPGTSASATLTGLTRGATYNIIVEAVKDQQRQKVREEVVTVGNSVDQGLSQPTDDSCFDPYTVSHYAIGEEWERLSDSGFKLSCQCLGFGSGHFRCDSSKWCHDNGVNYKIGEKWDRQGENGQMMSCTCLGNGKGEFKCDPHEATCYDDGKTYHVGEQWQKEYLGAICSCTCFGGQRGWRCDNCRRPGAEPGNEGSTAHSYNQYSQRYHQRTNTNVNCPIECFMPLDVQADREDSRE
Sequence Assay Result Units