Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type.

Abstract

Mutations D187N and D187Y in domain 2 of the actin-regulating protein gelsolin cause familial amyloidosis-Finnish type (FAF). We have constructed and expressed a recombinant version of gelsolin domain 2 that is sufficiently stable for kinetic and equilibrium measurements. The wild-type domain and the two amyloidogenic mutants fold via simple two-state kinetics without the accumulation of an intermediate. Unfolding kinetics exhibits significant curvature with increasing urea concentration, indicating that the transition state for unfolding becomes more native-like under increasingly denaturing conditions in accordance with the Hammond postulate. Mutations D187N and D187Y destabilize gelsolin domain 2 by 1.22 and 2.16 kcal. mol(-1) (1 kcal = 4.18 kJ) respectively. The mutations do not prevent disulfide bond formation despite their direct contiguity with a cysteine residue involved in disulfide linkage. The destabilization conferred on gelsolin domain 2 by the FAF mutations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis at body temperature. Study holds ProTherm entries: 6490, 6491, 6492, 6493 Extra Details: protein; stability; denaturation; Hammond; two-state

Submission Details

ID: ezWdA95J

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:32 p.m.

Version: 1

Publication Details
Isaacson RL;Weeds AG;Fersht AR,Proc. Natl. Acad. Sci. U.S.A. (1999) Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type. PMID:10500162
Additional Information

Study Summary

Number of data points 12
Proteins Major actin ; Gelsolin
Unique complexes 3
Assays/Quantities/Protocols Experimental Assay: Cm temp:15.0 C ; Experimental Assay: m temp:15.0 C ; Experimental Assay: dG_H2O temp:15.0 C ; Experimental Assay: Cm temp:25.0 C ; Experimental Assay: m temp:25.0 C ; Experimental Assay: dG_H2O temp:25.0 C
Libraries Mutations for sequence A:DGEDVQALVIDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHTGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLNPKANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVSHTVPIYEGYALPHAILRLDLAGRDLTDYMMKILTERGYSFTTTAEREIVRDIKEKLAYVALDFEQEMATAASSSALEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESAGIHETTYNSIMKCDVDIRKDLYGNVVLSGGTTMFPGIADRMNKELTALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKEEYDESGPSIVHRKCF/G:MVVEHPEFLKAGKEPGLQIWRVEKFDLVPVPTNLYGDFFTGDAYVILKTVQLRNGNLQYDLHYWLGNECSQDESGAAAIFTVQLDDYLNGRAVQHREVQGFESATFLGYFKSGLKYKKGGVASGF

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3J0S 2011-11-24T00:00:00+0000 9.0 Remodeling of actin filaments by ADF cofilin proteins
4EFH 2012-03-29T00:00:00+0000 2.48 Acanthamoeba Actin complex with Spir domain D
6TF9 2019-11-13T00:00:00+0000 4.8 Structure of the vertebrate gamma-Tubulin Ring Complex
1D0N 1999-09-13T00:00:00+0000 2.5 THE CRYSTAL STRUCTURE OF CALCIUM-FREE EQUINE PLASMA GELSOLIN.
1RGI 2003-11-12T00:00:00+0000 3.0 Crystal structure of gelsolin domains G1-G3 bound to actin
2FGH 2005-12-22T00:00:00+0000 2.8 ATP bound gelsolin
1HLU 1997-05-30T00:00:00+0000 2.65 STRUCTURE OF BOVINE BETA-ACTIN-PROFILIN COMPLEX WITH ACTIN BOUND ATP PHOSPHATES SOLVENT ACCESSIBLE
2BTF 1994-01-18T00:00:00+0000 2.55 THE STRUCTURE OF CRYSTALLINE PROFILIN-BETA-ACTIN
2OAN 2006-12-16T00:00:00+0000 2.61 Structure of oxidized beta-actin
3U4L 2011-10-09T00:00:00+0000 2.4 Cryocooled bovine profilin:actin crystal structure to 2.4 A

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 A Major actin P07830 ACT1_DICDI
99.2 A Major actin Q54GX7 ACT10_DICDI
98.9 A Major actin P02576 ACTA_PHYPO
98.4 A Major actin Q553U6 ACT22_DICDI
97.6 A Major actin P02578 ACT1_ACACA
96.5 A Major actin P07829 ACT3_DICDI
95.7 A Major actin O93400 ACTB_XENLA
95.7 A Major actin Q6NVA9 ACTB_XENTR
95.7 A Major actin P10987 ACT1_DROME
95.7 A Major actin P84184 ACT3B_HELAM
95.7 A Major actin P84183 ACT4_BOMMO
95.7 A Major actin P84185 ACT5C_ANOGA
95.2 A Major actin P10984 ACT2_CAEEL
95.2 A Major actin P53471 ACT2_SCHMA
95.7 A Major actin P53505 ACT5_XENLA
95.4 A Major actin P60712 ACTB_BOVIN
95.4 A Major actin O18840 ACTB_CANLF
95.4 A Major actin Q71FK5 ACTB_CAVPO
95.4 A Major actin P60706 ACTB_CHICK
95.4 A Major actin Q76N69 ACTB_CHLAE
95.4 A Major actin P60708 ACTB_HORSE
95.4 A Major actin P60709 ACTB_HUMAN
95.4 A Major actin Q4R561 ACTB_MACFA
95.4 A Major actin Q711N9 ACTB_MESAU
95.4 A Major actin P60710 ACTB_MOUSE
95.4 A Major actin Q5R1X3 ACTB_PANTR
95.4 A Major actin Q6QAQ1 ACTB_PIG
95.4 A Major actin Q5R6G0 ACTB_PONAB
95.4 A Major actin P60711 ACTB_RAT
95.4 A Major actin P60713 ACTB_SHEEP
95.4 A Major actin Q4L0Y2 ACTB_SPECI
95.4 A Major actin P60707 ACTB_TRIVU
95.7 A Major actin P63258 ACTG_BOVIN
95.7 A Major actin Q5ZMQ2 ACTG_CHICK
95.7 A Major actin P63261 ACTG_HUMAN
95.7 A Major actin P63260 ACTG_MOUSE
95.7 A Major actin Q5JAK2 ACTG_PELLE
95.7 A Major actin P63259 ACTG_RAT
95.7 A Major actin P63257 ACTG_TRIVU
95.7 A Major actin A2BDB0 ACTG_XENLA
95.7 A Major actin Q25010 ACT3A_HELAM
95.2 A Major actin Q93129 ACTC_BRABE
95.4 A Major actin Q6P378 ACTG_XENTR
95.2 A Major actin P41340 ACT3_LIMPO
95.4 A Major actin P18603 ACT4_ARTSX
96.0 A Major actin P12716 ACTC_PISOC
94.9 A Major actin P30162 ACT1_ONCVO
95.2 A Major actin P02572 ACT2_DROME
95.2 A Major actin P53478 ACT5_CHICK
95.2 A Major actin P48975 ACTB_CRIGR
95.2 A Major actin P30163 ACT2_ONCVO
95.2 A Major actin P68142 ACTB1_TAKRU
95.2 A Major actin P53486 ACTB3_TAKRU
95.2 A Major actin P68143 ACTB_OREMO
95.2 A Major actin Q91ZK5 ACTB_SIGHI
94.9 A Major actin Q93131 ACTC_BRAFL
95.2 A Major actin Q8JJB8 ACTG_TRISC
94.6 A Major actin P10986 ACT4_CAEEL
94.9 A Major actin P79818 ACTB_ORYLA
95.4 A Major actin P15475 ACTB_XENBO
95.4 A Major actin P04829 ACT3_BOMMO
94.4 A Major actin P0DM41 ACT1_CAEEL
94.4 A Major actin P0DM42 ACT3_CAEEL
95.4 A Major actin P53506 ACT8_XENLA
94.9 A Major actin O42161 ACTB_SALSA
94.9 A Major actin Q7ZVI7 ACTB1_DANRE
95.2 A Major actin P29751 ACTB_RABIT
95.2 A Major actin Q26065 ACT_PLAMG
94.4 A Major actin O17320 ACT_CRAGI
94.6 A Major actin Q7ZVF9 ACTB2_DANRE
94.6 A Major actin P83751 ACTB_CTEID
94.6 A Major actin P83750 ACTB_CYPCA
94.9 A Major actin Q964E1 ACTC_BIOOB
94.9 A Major actin Q964E2 ACTC_BIOPF
94.4 A Major actin O18499 ACT1_SACKO
94.9 A Major actin P53470 ACT1_SCHMA
94.6 A Major actin Q964E3 ACTC_BIOAL
95.2 A Major actin P63256 ACTG_ANSAN
94.4 A Major actin P53485 ACTB2_TAKRU
94.9 A Major actin P53461 ACTC_HALRO
94.6 A Major actin Q0PGG4 ACTB_BOSMU
94.6 A Major actin P92179 ACTC_BIOGL
94.4 A Major actin Q964E0 ACTC_BIOTE
94.4 A Major actin Q964D9 ACTC_PLATR
94.9 A Major actin P90689 ACT_BRUMA
94.9 A Major actin P84336 ACTB_CAMDR
94.4 A Major actin P17126 ACT_HYDVU
94.6 A Major actin P41339 ACTA_LIMPO
94.1 A Major actin P49871 ACT_MANSE
93.8 A Major actin P07836 ACT1_BOMMO
93.8 A Major actin P68556 ACT1_DIPDE
94.9 A Major actin P41112 ACT1_PODCA
93.8 A Major actin P68555 ACT_TAESO
93.6 A Major actin P45886 ACT3_BACDO
94.1 A Major actin P41113 ACT3_PODCA
93.6 A Major actin P45885 ACT2_BACDO
93.6 A Major actin P92182 ACT1_LUMTE
93.6 A Major actin O17503 ACTC_BRALA
93.6 A Major actin P53456 ACT2_DIPDE
93.3 A Major actin P53501 ACT3_DROME
94.1 A Major actin O18500 ACT2_SACKO
93.3 A Major actin P10981 ACT5_DROME
94.1 A Major actin P41341 ACTY_LIMPO
94.9 A Major actin P69002 ACT1_HELER
94.9 A Major actin P69003 ACT1_HELTB
94.3 A Major actin P53458 ACT5_DIPDE
93.3 A Major actin P18601 ACT2_ARTSX
94.1 A Major actin P92176 ACT2_LUMTE
93.3 A Major actin P53464 ACTM_HELTB
93.0 A Major actin O16808 ACT_MAYDE
93.8 A Major actin P83969 ACT1_BACDO
93.8 A Major actin P83967 ACT6_DROME
93.8 A Major actin P83968 ACT6_DROSI
94.6 A Major actin P53472 ACTA_STRPU
94.1 A Major actin P17304 ACTM_APLCA
92.8 A Major actin P18600 ACT1_ARTSX
93.3 A Major actin P53463 ACTM_HELER
93.3 A Major actin P07837 ACT2_BOMMO
93.3 A Major actin P49128 ACT1_AEDAE
93.0 A Major actin P53457 ACT3_DIPDE
93.3 A Major actin P12717 ACTM_PISOC
93.3 A Major actin P91754 ACT_LUMRU
94.6 A Major actin P69004 ACT2_MESFR
94.6 A Major actin P69005 ACTD_STRPU
91.7 A Major actin P53496 ACT11_ARATH
94.6 A Major actin P10990 ACT1_MESFR
91.5 A Major actin A2XLF2 ACT1_ORYSI
91.5 A Major actin Q10DV7 ACT1_ORYSJ
94.4 A Major actin P53473 ACTB_STRPU
92.3 A Major actin O65315 ACT_COLSC
93.6 A Major actin P53465 ACT1_LYTPI
92.8 A Major actin P45887 ACT5_BACDO
92.5 A Major actin P62739 ACTA_BOVIN
92.5 A Major actin P62736 ACTA_HUMAN
92.5 A Major actin P62737 ACTA_MOUSE
92.5 A Major actin P62740 ACTA_RABIT
92.5 A Major actin P62738 ACTA_RAT
90.9 A Major actin Q05214 ACT1_TOBAC
92.2 A Major actin Q25472 ACT2_MOLOC
90.9 A Major actin P30167 ACT3_SOLTU
92.5 A Major actin P02574 ACT4_DROME
92.2 A Major actin P08023 ACTA_CHICK
92.0 A Major actin Q3ZC07 ACTC_BOVIN
92.0 A Major actin P68034 ACTC_CHICK
92.0 A Major actin P68032 ACTC_HUMAN
92.0 A Major actin P68033 ACTC_MOUSE
92.0 A Major actin P68035 ACTC_RAT
92.0 A Major actin Q6P640 ACTC_XENTR
92.2 A Major actin Q5E9B5 ACTH_BOVIN
92.2 A Major actin P63270 ACTH_CHICK
92.2 A Major actin P63267 ACTH_HUMAN
92.2 A Major actin P63268 ACTH_MOUSE
92.2 A Major actin P63269 ACTH_RAT
90.9 A Major actin O81221 ACT_GOSHI
91.7 A Major actin P20399 ACT2_XENTR
91.2 A Major actin A2XNS1 ACT3_ORYSI
91.2 A Major actin Q10AZ4 ACT3_ORYSJ
93.6 A Major actin Q07903 ACTC_STRPU
92.0 A Major actin P04751 ACTC_XENLA
90.7 A Major actin P30171 ACT11_SOLTU
92.8 A Major actin P53466 ACT2_LYTPI
92.0 A Major actin P27130 ACT2_HALRO
91.7 A Major actin P04752 ACT3_XENLA
91.7 A Major actin Q6P8G3 ACT3_XENTR
91.7 A Major actin P53459 ACT6_DIPDE
92.8 A Major actin Q00215 ACTC_STYPL
92.2 A Major actin O15998 ACTM_CIOSA
90.4 A Major actin P0CJ46 ACT1_ARATH
90.4 A Major actin P0CJ47 ACT3_ARATH
91.2 A Major actin P53492 ACT7_ARATH
91.5 A Major actin P53480 ACTC_TAKRU
92.8 A Major actin P18499 ACTF_STRPU
91.2 A Major actin P49055 ACTS_CARAU
93.0 A Major actin P53474 ACTE_STRPU
91.2 A Major actin P68138 ACTS_BOVIN
91.2 A Major actin P68139 ACTS_CHICK
91.2 A Major actin P68133 ACTS_HUMAN
91.2 A Major actin P68134 ACTS_MOUSE
91.2 A Major actin Q98972 ACTS_ORYLA
91.2 A Major actin P68137 ACTS_PIG
91.2 A Major actin Q5R9Q5 ACTS_PONAB
91.2 A Major actin P68135 ACTS_RABIT
91.2 A Major actin P68136 ACTS_RAT
91.2 A Major actin P10995 ACT2_XENLA
91.8 A Major actin P53475 ACTN_STYCL
91.2 A Major actin P53482 ACTSB_TAKRU
90.9 A Major actin P68140 ACTSA_TAKRU
90.9 A Major actin P68264 ACTS_OREMO
90.6 A Major actin P26197 ACT2_ABSGL
90.9 A Major actin Q90X97 ACTS_ATRMM
90.7 A Major actin P30173 ACT13_SOLTU
90.7 A Major actin P53479 ACTS_CYPCA
91.2 A Major actin P53460 ACT1_HALRO
90.1 A Major actin P53494 ACT4_ARATH
91.5 A Major actin P26198 ACTM_STYCL
91.4 A Major actin P53689 ACT_PHARH
90.7 A Major actin P53498 ACT_CHLRE
91.4 A Major actin P20904 ACT_VOLCA
90.1 A Major actin P53504 ACT1_SORBI
91.2 A Major actin Q9Y707 ACT2_SUIBO
90.6 A Major actin P50138 ACT_PUCGR
95.6 A Major actin P84856 ACTB_CHLPG
97.2 A Major actin Q55EU6 ACT23_DICDI
90.1 A Major actin P53467 ACTM_MOLOC
90.3 A Major actin Q9Y702 ACT1_SCHCO
90.4 A Major actin P30164 ACT1_PEA
90.3 A Major actin Q00214 ACTM_STYPL
90.6 A Major actin P12431 ACTM_STRPU
90.1 A Major actin Q9Y701 ACT1_SUIBO
92.2 A Major actin P30172 ACT12_SOLTU
92.3 A Major actin P81228 ACT5_SOLTU
92.0 A Major actin P81229 ACT8_SOLTU
92.0 A Major actin P93584 ACT9_SOLTU
92.0 A Major actin P93372 ACT4_TOBAC
94.5 A Major actin P18602 ACT3_ARTSX
91.1 A Major actin Q96482 ACT1_SOLLC
91.4 A Major actin Q96484 ACT3_SOLLC
91.4 A Major actin P93371 ACT5_TOBAC
92.0 A Major actin P93375 ACT7_TOBAC
91.4 A Major actin Q96483 ACT2_SOLLC
91.1 A Major actin P93587 ACT1_SOLTU
91.1 A Major actin P93376 ACT6_TOBAC
90.5 A Major actin P93585 ACT4_SOLTU
92.6 A Major actin Q03342 ACT3_ECHGR
91.7 A Major actin Q92192 ACT_CALFI
94.0 A Major actin Q92193 ACT_CRAVI
96.9 A Major actin P30170 ACT10_SOLTU
97.0 A Major actin Q11212 ACT_SPOLI
90.1 A Major actin Q25381 ACTM_LYTPI
91.3 A Major actin Q25379 ACT3_LYTPI
92.6 A Major actin P10982 ACT1_ABSGL
95.5 A Major actin P00544 FGR_FSVGR
90.4 A Major actin P10994 ACTS_PLEWA
97.6 A Major actin Q39596 ACT_OXYRB
97.8 A Major actin P85911 ACT1_PSEMZ
96.9 A Major actin Q24733 ACT_DICVI
100.0 G Major actin P06396 GELS_HUMAN
99.2 G Major actin Q3SX14 GELS_BOVIN
99.2 G Major actin P20305 GELS_PIG
98.4 G Major actin Q28372 GELS_HORSE
97.6 G Major actin P13020 GELS_MOUSE
97.6 G Major actin Q68FP1 GELS_RAT