DISEÑO DE CONEXIONES

If sequence differences betw een opsins m ediate ^max/ the next questions to arise are w hat are these differences and w here do they occur w ithin the opsin? C om parison of the am ino acid sequences of different classes of o p sin s m ay h ig h lig h t the resid u es resp o n sib le for these variations in ^max-

As m entioned previously (Section 1.14), all the m em bers of the L group of pigm ents contain two conserved positively charged residues, His- 181 and Lys-184. These two residues bind chloride ions (Cl") resulting in a

shift of Im ax to longer w avelengths (W ang et ah, 1993). The Xmax of

h u m an red and green cone pigm ents are shifted from 535nm an d 515nm in a Cl" depleted environm ent to 560nm and 530nm u p o n ad d itio n of Cl",

respectively. 560nm and 530nm are the approxim ate v alu es of Xmax

obtained by m icrospectrophotom etry of single h u m an red and green cone

cells, respectively (Dartnall et ah, 1983). Thus, chloride ions w ithin the L

g roup of cone cells play a role in spectral tu n in g and this in tu rn is dependent on the presence of the residues His-181 and Lys-184 w ithin the opsin. All other vertebrate opsins outside of the L group (S /M l/M 2 /R h ) contain Glu-181 and Gln-184 (except the goldfish u ltra v io le t p ig m en t w hich contains Gly-184). A lthough this explains how the L g ro u p of opsins attain red shifts in their ^max in relation to the other visual groups, this does n o t explain the differences in Xmax betw een in d iv id u al m em bers

of the L group. For exam ple, w hy is there a 30nm difference in ^max betw een the hum an red and green cone pigments?

The nucleotide sequences of the h u m an red cone p ig m en t (RCF) an d g reen cone p ig m en t (GCP) genes are 98% id en tical, an d th eir respective p ro tein s only differ at 15 out of 364 am ino acid resid u es

(N athans et al., 1986a). The 30nm red shift from the GCP to the RCP w as

initially th o u g h t to be attributed to the substitution of non-polar residues w ith hydroxyl-bearing amino acids at only three of these 15 sites, site 164,

261 and 269, (site num bering using the bovine rod opsin system ) (Neitz et

al., 1991). A n a d d itio n a l p o sitio n , site 217, w h ich also involves

su b stitu tio n of a hydroxyl-bearing am ino acid for a n o n p o lar residue betw een the hu m an RCP and GCP, w as im plicated from studies on N ew an d O ld W orld m onkey visual p ig m en ts b elo n g in g to th e L g ro u p

(Ibbotson et al., 1992; W illiams et al., 1992). Site-directed m utagenesis

stu d ies finally established th at su b stitu tio n at 7 of these 15 residues resulted in com plete conversion of the ^max observed in one pigm ent to

the other (Asenjo et ah, 1994). These seven spectral tu n in g sites and the

residues w ithin the hum an RCP and GCP at these positions are given in Table 3.1. Substitution at any of one these seven sites results in a shift of Xmax- Every extra replacem ent on top of the initial substitution produces an additive effect until the 30nm difference betw een the tw o pigm ents is com pensated for w hen all seven residues are substituted. Substitutions at sites 261 and 269 are attributed to causing tw o thirds of the 30nm shift (Asenjo et ah, 1994).

The hydroxyl groups of an amino acid are thought to interact w ith the chrom ophore either in a direct or indirect m anner to pro d u ce shifts in

^max (Chan et ah, 1992). In a sim ilar fashion, it w as hypothesised that an

electrostatic interaction betw een the Schiff base and charged am ino acid residues w ithin the opsin could cause shifts in Xmax (Kropf an d H ubbard, 1958). This is true for the charged residue Clu-113, the counter-ion to the p ro to n ated Schiff base, w hich influences spectral tu n in g by electrostatic

interaction w ith the chrom ophore (Sakmar et ah, 1989; Z hukovsky and

O prian, 1989). In the search for the counter-ion another spectral tu n in g site w as discovered, nam ely site 122. Substitution of Clu-122 to Cln-122 or Ala-122, in bovine rod opsin, caused a blue shift in Xmax d o w n from 498nm to 482nm and 476nm , respectively (Nakayama and K horana, 1991). Both Ala and Gin are uncharged residues. Thus, these non-conserved substitutions for the charged Clu residue at site 122 result in shifts of Xmax-

Am ino acid position RCP GCP 1 0 0 Ser Tyr 164 Ser A la 214 lie T h r 217 Ala Ser 261 Tyr Phe 269 T h r Ala 293 Tyr Phe

T able 3.1 Seven amino acid sites w ith their respective residues w ithin the h u m an red cone pigm ent (RCP) and green cone pigm ent (GCP). Exchange of residues at these sites betw een the RCP and GCP result in shifts of ^max

(Asenjo et ah, 1994). Replacement of all these 7 residues found at the same

site in the GCP for the resid u es in the RCP resu lts in a com plete

Both G lu a n d A sp resid u es are n eg ativ ely ch arg ed a m in o acids. Substitution of Glu-122 for Asp-122 is considered a conservative change, in spectral tu n in g term s. H ow ever, substitution of Glu-122 for Asp-122, in bovine rod opsin, also gives a Xmax of 476nm (N akayam a and K horana, 1991). Thus, the spectral tuning effect at site 122 is thought to be a steric one rather than an interaction of electrostatic charge.

Thus, it w ould seem that conservative am ino acid substitutions can affect the Xmax of a v isu al p ig m en t, as w ell as n o n -co n serv a tiv e replacem ents, d ep en d in g on the position of the am ino acid w ith in the opsin.